RNA Processing: A Practical Approach: Volume II

RNA Processing ,\ Practical Approach \'OJ.l i:\IE II

Edited by S. J. HI GGINS a nd B. D. HAM ES

7

u

l1

t

mG

•GTP

3'

5'

I

(I

I + *G '011

AAUAAA

r-(A)0

~

- -

RNA Processing Voluine II A Practical Approach Edited by STE P H E N J. HIG GI N S and B . DA VI D H A ME S Department of Biochemistry and Molecular Biology, University of Leeds

-atOXFORD l'XT\'ERSITY PRESS

Oxford :'lit:w York Tokyo

llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll

~t

The Practical Approach Series SERIES EDITORS

D. RICKWOOD Deporrment of B1ology, Uni~·ersuy of Essex Wn·enhoe Pork, Colchester, Essex C04 JSQ, UK

B. D. HAMES Deportment of Biochemistr> and ..,1oleculor Btolog; Unn·ersity of Leeds, Leeds LS2 9JT, UK

Affinity Chromatography Anaerobic Microbiology Animal Cell Culture (2nd edition) Animal Virus Pathogenesis Antibodies I and II Behavioural Neuroscience Biochemical Toxicology Biological Data Analysis Biological Membranes Biomechanics-Materials Biomechanics-Structures and Systems Biosensors Carbohydrate Analysis Cell-Cell Interactions The Cell Cycle Cell Growth and Division Cellular Calcium Cellular Interactions in Development Cellular Neurobiology Centrifugation (2nd edition) Clinical Immunology Computers in Microbiology Crystallization of Nucleic Acids and Proteins Cytokines The Cytoskeleton Diagnostic Molecular Pathology I and II Directed \.iutagenesis

DNA Cloning I, 11, and £II Drosophila Electron Microscopy m Biology Electron Microscopy in Molecular Biology Electrophysiology Enzyme Assays Essential Developmental Biology Essential Molecular Biology I and II Experimental Neuroanatomy Fermentation Flow Cytometry Gas Chromatography Gel Electrophoresis of Nucleic Acids (2nd edition) Gel Electrophoresis of Proteins (2nd edition) Gene Targeting Gene Transcription Genome Analysis Glycobiology Growth Factors Haemopoiesis Histocompatibility Testing HPLC of Macromolecules HPLC of Small Molecules Human Cytogenetics r and II (2nd edition) Human Genetic Disease Analysis

Immobilized Cells and Enzymes Immunocytochemistry In Situ Hybridization Iodinated Density Gradient Media Light \ticroscopy in Biolog} Lipid Analysis Lipid todification of Proteins Lipoprotein Analysis Liposomes Lymphocytes Mammalian Cell Biotechnology \1ammalian Development !\1edical Bacteriology !\1edical Mycology !\1icro..:omputers in Biochemistry Microcomputers in Biology Microcomputers in Physiology Mitochondria Molecular Genetic Analysis of Populations Molecular Imaging in Neuroscience Molecular Neurobiology ~tolecular Plant Pathology I and li Molecular Virology Monitoring Neuronal Activity Mutagenicity Testing "leural Transplantation Neurochemistry l'ieuronal Cell Lines NMR of Biological Macromolecules Nucleic. Acid and Protein Sequence Analysis Nucleic Acid Hybridisation Nucleic Acids Sequencing Oligonucleotides and Analogues Oligonucleotide Synthesis PCR Peptide Hormone Action

Peptide Hormone Secretion Photosynthesis: Energy Transduction Plant Cell Culture Plant Molecular Biology Plasmids (2nd edition) Pollination Ecolog:r Postimplantation 1\lammalian Embryos Preparative Centrifugation Prostaglandins and Related Substances Protein Architecture Protein Engineering Protein Function Protein Phosphorylation Protein Purification Applications Protein Purification Methods Protein Sequencing Protein Structure Protein Targeting Proteolytic Enzymes Radioisotopes in Biology Receptor Biochemistry Receptor-Effector Coupling Receptor-Ligand Interactions Ribosomes and Protein Synthesis RNA Processing Signal Transduction Solid Phase Peptide Synthesis Spectrophotometry and Spectrofluorimetry Steroid Hormones Teratocarcinomas and Embryonic Stem Cells Transcription Factors Transcription and Translation Tumour lmmunobiology Virolog) Yeast

Oxjord L'nn·ersit.> Press, Walton Street, Oxford OXl 6DP Oxford New York Toronto De/111 Bombay Calcuua Madras /wrach1 Petalmg Jaya Singapore Hong Kong Tokyo 'liairobt Dar es Salaam Cape Town \felbourne Auckland and associated companies in Berlm lbadan Oxford

IS

a trade mark of Oxford Umversu; Press

(b

A Pract1ca/ Approach is a regtstered trade mark of the Chancellor, \fosters, and Scholars of the Unmm1ty of Oxford trading as Oxford Universtty Press Published in the United States by Oxford Um>oersuy Press, New York @Ox/i>rd Uniwrw\ Press. /994 All rights reserved. !\'o part of thts pub/teal/on may be reproduced, stored m a reme•·a/ system. or transmmed, in any form or by any means, >wthout the prior permiSSIOn In wrtllng of Oxford Unll•ersity Press. Within the UK, exceptions are allowed in respect of any fatr dealing for the purpose of research or private study, or criticism or rewew, as permuted under the Copyright, Designs and Patents Act, 1988, or in the case of reprographic reproduction in accordance w1th the terms of l1cences issued by the Copyright Licensmg Agency. Enquiries concerning reproduction outside those terms and m other countries should be sent to the Rtghts Department, Oxford Uni,·erslty Press, at the address above. This book 1s sold subJect to the conditton that 11 shall not, by way of trade or otherwise, be lelll, re-sold, hired out, or otherwise ctrculated wuhout the publisher's prior consent in an; form of binding or cover other than that In which 11 ts published and without a similar condition including this condition being tmposed on the subsequent purchaser. Users of books tn the Practical Approach Series are advised that prudent laboratory safety procedures should be followed at all times. Oxford Universuy Press makes no representation, express or 11nplied, m respect of the accuracy of the material set forth In books in tl11s sertes and cannot accept any legal responsibility or liability for any errors or otmssions that may be made. A catalogue record for tillS book is available from the Brittsh Library Ltbrary of Congress Cataloging m Publication Data RNA processmg: o practical approach edited by Stephen J. H1ggins and B. David Hames. p. cm.-(The Practical approach series, /35, 136) Includes b1blwgraphical references and mdex. I. RI\A-Research-l..oborotory manuals. 1. GenetiC regulotionResearch-I.Aborotory manuals. I. Higgins, S. J. (Ste,·e J.J f/ Homes, 8. D. Iff. Series QP623.R58 !993 574.87'3283-dclO 93-13019 ISBN 0 19 963344 4 (hlb) ISBN 0 19 963343 6 (plb) ISBN 0 19 963471 8 fhlb) ISBN 0 /9 963470 X (p/b)

J Volume I

J

Volume II

Typeset by Dobb1e Typesetlmg Ltmued, Tavwock, Devon Prmted and bound m Great Bmom by Information Press, Ltd.. Eynsham, Oxon

Foreword RNA processing: two helpful guides for cutting , pasting, trimming , and editing RNA TOM MANIATIS

During the past decade, the study of RNA processing has risen to the forefront of molecular biology. At one time, the RNA processing field was a relatively small group of investigators interested in the trimming and packaging of prokaryotic transfer RNA and ribosomal RNAs. However, with the discovery of a bewildering array of RNA processing events in both prokaryotes and eukaryotes, the field has become a burgeoning enterprise. This change is reflected in the annual RNA processing meeting which has been rapidly transformed from a small informal gathering to one bursting the seams of the expanded meeting facilities at the Cold Spring Harbor Laboratory. Due to the pace of this unprecedented growth in size and diversity, no one has stopped long enough to compile a detailed description of even the most basic techniques used to study RNA. Students in the field are therefore introduced to the laboratory with handwritten protocols passed from person to person. The methods compiled in this book and its companion volume by leaders in the field should therefore contribute significantly to the training of new students and hopefully lead to the development of new techniques. Volumes I and II of RNA processing: a practical approach cover virtually all aspects of RNA processing, including capping, splicing of both pre-mRNA and tRNA, polyadenylation, editing and ribosomal RNA processing. In addition, an overvtew of ribozymes is provided. An understanding of the various types of RNA processing in vivo is an essential prerequisite for studying in vitro the mechanisms involved . Therefore, the description of methods for RNA mapping, including nuclease Sl mapping, primer extension analysis, and the use of the polymerase chain reaction should be very useful. An important technical advance in the analysis of RNA processing in vitro was the development of plasmid vectors containing bacteriophage-specific promoters for synthesizing labelled substrate RNAs. A description of the Synthesis and purification of in vitro transcripts produced with SP6, T7, and polymerase is therefore an essential part of the coverage. Also important lS a description of the preparation and optimization of nuclear extracts for several

!3

Foreword types of in vitro RNA processing, 3 '-end formation and polyadenylation. The manipulation and fractionation of these extracts have led to the purification of individual processing components and the cloning and characterization of the corresponding genes. Methods have also been developed for the selective inactivation of individual snRNPs using specific DNA oligonucleotides, or the depletion or punfication of )nRNP particles using oligo(2' -0-alkylribonucleotides). In addition, immunoaffin.ity purification methods for purifying snRNPs are presented as well as procedures for reconstituting individual snRNPs from purified proteins and RNAs. These tools for characterizing snRNPs will become increasingly important as the details of protein-protein and protein-RNA interactions in the spliceosome are unravelled . An area less intensely investigated but of increasing importance, is the turnover of mRNA. There are now many examples in which gene expression during development is regulated by the selective turnover of specific RNAs. In add:ition, it is now clear that the rapid decrease in the expression of cyrokines after induction by extracellular inducers involves a specific recognition sequence in the 3 non-coding sequence of mRNA. The chapter describing methods for studying mRNA turnover in vitro will stimulate further studies of this important problem. The most recently discovered and most unexpected forms of RNA processing are autocatalytic splicing and RNA editing. The discovery of group I intron self-splicing and the RNase P ribozyme has led rapidly to detailed characterization of the catalytic reactions involved. Dissection and manipulation of the group I intron ribozyme have led to the creation of new catalytic activities and an understanding of the role of RNA secondary and tertiary structure in ribozyme function . Similarly, the elucidation of the structure and activity of group li introns has provided important new insights into the nature of ribozymes and has led to critical insights into the mechanisms involved in pre-mRNA splicing. Both group 11 intron and pre-mRNA splicing proceed through a similar branched RNA intermediate. Although the former process is autocatalytic and the latter require~s multiple components assembled into a spliceosome, the catalytic events may be quite similar. In fact, the parallels between the role of RNA structure in group II intron splicing and the role of snRNA-pre-mR'JA interactions have increased by the recent demonstration of dynamic interacuons between snRI'
viii

Fore word pro ·e,~in:: The discovery o f guide RNAs complementary to edited poruons of mR \, -\ and the identification of putati' e intermediates in the editing processes have led o the proposal of a specific mechanism for RNA editing. This mechanism invo l\'es an orderly cycling of the editing process in a 3 to 5 direction. Specific catalytic mechanisms have been proposed based in part on comparisons to the group I and II self-splicing reactions. The detailed description in R 'A processing: a practical approach of the biological systems and techniques used to study RNA editing should stimulate the development of new approaches to the study of the mechanisms involved. Alt hough most of this two volume set focuses on the use of biochemical approaches to RNA processing, a description of genetic techniques used to study pre-mRNA processing in yeast is also provided. The application of these techmques has led to the identification and cloning of genes encoding essential splicing factors (PRP genes). In addition, a variety of genetic tools, including gene 'knockouts' , targeted mutagenesis, interactive suppression, and conditional expression vectors have been used to study the function of a number of yeast splicing components including PRP proteins and specific snRNAs. These tools have been used in conjunction with in vitro studies using nuclear extracts prepared from wild type yeast strains as well as strains lacking an essential splicing factor. Comparison of the results of these studies with those of mammalian splicing has revealed a remarkable conservation in the mechanisms of the splicing reaction and spliceosorne assembly, and in the role of snRNPs in splice site recognition. This comparison of yeast and mammalian splicing points to an additional benefit of collecting a variety of RNA processing methods together. It provides the opportunity to examine the similarities and differences in experimental approaches established for studying different types of RNA processing. Hopefully, this will lead to previously unrecognized connections and new ideas. 1

I

ix

Preface This book arose out of the success of a book we edited for the Practical Approach series in 1984, entitled Transcription and translation: a practical approach. When the time came to consider organizing a second edition, it rapid!) became clear that no one book of the desired size could include, in sufficient detail, the myriad of important new techniques, particularly in the area of posttranscriptional processing of RNA transcripts, that had arisen since the first edition. Thus the logical decision was taken to produce several books, the first of which, Gene transcription: a practical approach, has recently been published. R NA processing: a practical approach Volumes I and II are the next books in this planned set and are companion volumes. RNA processing Volume ri includes contributions from Walter Ketler's laboratory on techniques for analysis of 3' end-processing of mRNA and from Aaron Shatkin's group on capping and methylation of mRNA. RNA editing and the analysis mRNA turnover are also included in this volume, with chapters by Larry Simpson and his colleagues and Jeff Ross, respectively. Barbara Sollner-Webb and Cathy Enright describe methods for ribosomal RNA processing and Chris Greer similarly covers transfer RNA processing. Finally, David Shub, Craig Peebles, and Arnold Hampel have combined their efforts to produce a very topical chapter covering investigations of detailed splicing reactions, comprehensively covering group I intron slicing, group lJ intron splicing and hammerhead and hairpin ribozymes. The companion volume, RNA processing Volume I, begins with Benoit Chabot describing the synthesis and purification of RNA substrates fo r RNA processing investigations. Paula Grabowski then covers essential methods for the identification and analysis of spliced mRNAs. Next, Ian Eperon and Adrian Krainer have collaborated to produce an important chapter on the analysis of the splicing of mRNA precursors in mammalian cells. Another key area, the analysis of RNP complexes and their interactions, is covered in two chapters by Angus Lamond and Brian Sproat and by Reinhard Liihrmann and his colleagues. These are all central issues in RNA processing studies. Final I}. Andrew Ne\\ man has contributed a chapter on investigations of pre-mRNA splicing in yeast, which allows some approaches that are not possible with mammalian cells, most notably the isolation and analysis of splicing mutants. The aim of both books remains the aim of this popular series; to present, in a clear readable manner, the background to the range of techniques and experimental approaches available, to describe in precise detail a key selection of tried and tested protocols, and to discuss potential pitfalls, data interpretation, and a variety of other hints and tips for the active scientist. It is a measure of the efforts of our contributors that we believe these aims have beoen more than

Preface met in each volume. We thank them for their diligence in writing texts which address these aims and, where we felt that editorial changes were essential, for graciously accepting these. We hope and believe that the end result will be seen as a comprehensive and valuable two volume compendium of the best of current methodology in this subject area. Because of the scientific quality of the contributions and the deliberately explanatory style of writing, we are confident that, like their predecessor, these books will rightfully enjoy popularity among both newcomers to the field and more experienced researchers. Leeds February 1993

Stephen Higgins David Hames

xii

Contents

List of contributors Abbreviations

xix xxi

1. 3' end-processing of mRNA £/mar Wahle and Walter Keller 1 . Introduction: overview of 3' end-processing in manunalian cells

2. 3'-en d cleavage and polyadenylation in nuclear extracts Introduction RNA substrates for 3 ' end-processing in vitro Preparation of nuclear extracts for 3' end-processing Formation of polyadenylation complexes Assay of cleavage and polyadenylation in nuclear extracts

2 2 2 4 6 21

3. Polyadenylation and 3'-cleavage factors Overview of processing factors Purification of processing factors

26 26 26

4. Analysis of polyadenylation in vivo

33

References

2. Capping and methylation of mRNA

33 35

Yasuhiro Furuichi and Aaron J. Shatkin 1 . Introduction

35

2. Synthesis of capped mRNAs during in vitro transcription of viral

genes Introduction Strategy Synthesis of capped mRNAs using insect cytoplasmic polyhedrosis virus Synthesis of capped mRNAs using reovirus Synthesis of capped mRNAs using vaccinia vi rus In vitro transcription and capping of cellular mRNAs using bacteriophage RNA polymerases

38 38 38 39 41 42 43

Contents 3. Site-specific radiolabelling of caps in cellular mRNAs lntroducuon Rad•olabelling mR..'IAs by decapping and recapping Labelling of mRNA by periodate oxidation follo\\ed by reduction \\ilh [ lH ] sodium borohydride Labelling of mRNA by 2' -0-methylalion

45 45 45 50 51

4. Characterization of caps Strategy Enzymic analysis of caps Analysis of caps following enzyrmc d1gesuon

52 52 54 57

Acknowledgements

65

References

65

3. RNA editing in mitochondria

69

Larry Simpson, Agda M. Simpson, and Beat Blum 1. lntroduction PosHranscriplional modifications of eukaryotic mRNAs RNA editing in kineroplastid protozoa

69 69 69

2. Growth and maintenance of kinetoplastid protozoa

71 71

Choice of species for experimental work Growth and maintenance of L. rarenro/ae Cloning of kinetoplastid protozoan stock cultures Growth of cultures for production of kinetoplast components

3. Kinetoplast DNA Int roduction Isolation of kDNA Schizodeme-typing of kinetoplastid protozoan strains Isolation of maxicircle DNA

72 74 74

75 75 76 78

79

4. Isolation of the kinetoplast-mitochondrion

81

5. Isolation of transcripts from the mitochondrial genome

85 85

Introduction and strategy Isolation of kinetoplast RNA Northern blot analysis of kRNA Hybrid selection of guide RNAs PCR amplification of partially-edited mRNAs and gRNA:mR'IA chimaeric molecules Synthesis of gRNAs by transcription in l'itro

8i

88 88 90

91

6. Run-on transcription in isolated kinetoplast mitochondria

91

7. Identification of gRNAs by computer-assisted sequence comparison

94

xiv

Contents 8. Enzymatic activities in the kinetoplast-mitochondrion fraction which are involved in RI>.JA editing Introduction Preparation of mitochondrial extracts Terminal uridylyl transferase (TUTase) RNA ligase Cryptic R ase gR A:mRNA ch•maera-forrning activity

96 96

97 97 99 100 101

Acknowledgement

103

References

I03

4. Analysis of messenger RNA turnover in cell-free

extracts from mammalian cells

107

Jeff Ross 1. Introduction

107

2. Choice of mRl'JA substrate

I08 I09 J J0

Endogenous substrates (free mR P or polysome-associated mRNP) Exogenous substrates

3. Preparation of undegraded mRNA and polysomes

110

4. Preparation of cell extracts for in vitro mRNA decay reactions

III

5. Methods for performing in vitro mRNA decay reactions

119 119 123 125

Detergent-free extracts Lysolecithin extracts Reticulocyte translation extracts

6. mRNA detection, data interpretation, and troubleshooting mRNA detection and some typical in vitro mRNA decay experiments Data interpretation Troubleshooting

References

126 126 130 131 132

5. Ribosomal RNA processing in vertebrates

135

Cathleen Enright and Barbara Sol/ner- Webb 1. Introduction

135

2. Analysis of rR.'JA processing in vivo

137 137

Introduction Analysis of rRNA processing by hybrid selection and gel electrophoresis of cellular rRNAs Use of psoralen cross-linking for the analysis of associations bel\veen rRNAs in vivo Electron microscope analysis of rRNA synthesis (Miller spreading) XV

139 142 143

Contents

3. Analysis of rRNA processing in \'itro

144

lmroducuon Preparation of synthetic rR!-.A ~ubstrates Preparation and use of mouse S-100 processing extract Preparation and use of nucleolar and cytoplasmic processing extracts from HeLa cells Cleavage of pre-rRNA using purified nucleolar endonuclease Use of deletion analysis in the investigation of rRNA processing

144 144 148

4. Analysis of protein-RNA associations in rRNA processing Gel-shift analysis Analysis of rRNA associations by rate zonal centrifugation Use of UV cross-linking to detect polypeptides associated with rRNA in processing complexes

155 157 157

5. Assessment of the requirement for cellular R.J"'JA in rRNA processing Micrococcal nuclease digestion Oligonucleoude-directed RNase H digesuon of cellular R A~ in\olved m pre-rRNA processing Immunological analysis of polypeptides required for processing

150 155 155

160

164 164 165 169

6. rRNA processing associated with transcriptional termination 169 3' end-processing in the mouse 3' end-processing in Xenopus laevis

170 170

Acknowledgements

170

References

170

6. Processing of transfer RNA precursors

173

Chris L. Greer 1 . Introduction General methods tRNA structure and function Overview of processing pathways Biochemical analysis of tRNA proce~sing

173 173 173 175 176

2. Preparation of pre-tRNA processing substrates

176 176 176

Introduction Preparation of nuclease-free carriers Preparation of tRNA substrates from whole cells Preparation of substrates by in vitro transcription

3. Preparation of tRNA processing extracts Preparation of crude ceLl extracts Partial purification of processing activities Preparation of highly-purified processing components

xvi

177 181 185 187 189 191

Contents 4. Pre-tRNA processing assays Strategy Assay of pre-tRJ'IA splicing As a:r of base modifications and tR:-IA splice junctions

195 195 196 199

Acknowledgements

206

References

206

7 . Ribozymes

211

David A. Shub, Craig L. Peebles, and Arnold Hampel 1. Introduction

211

2. Group 1 intron ribozymes The self-splicing activity of group I introns Detection of self-splicing introns in cellular RNA Confirmation of self-splicing by in vitro transcription of cloned DNA containing putative group I introns

211 211 213

3. Group II intron ribozymes Identification of group l1 introns Group II intron secondary structure and functional anatomy Potential of group II intron ribozymes for practical use Self-splicing of group l1 introns Other reactions of group 11 intron ribozymes

217

217 219 220

4. Hammerhead and hairpin ribozymes

230

215

221

224

Introduction 230 Applications of hammerhead and hairpin ribozymes in molecular biology 232 Use of hammerhead and hairpin ribozymes to target specific RNA transcripts 232 Use of autocatalytic cassettes to generate transcripts with defined 5' or 3' termini 236

Acknowledgements

237

References

237

Appendix: Suppliers of specialist items Contents of Volume I Index

xvii

241

245 247

Contributors BEAT BLUM Department of Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland. CATHLEEN ENRIGHT Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA. Y AS UH lR O FURUICH I Biotechnology Subdivision, Nippon Roche K.K., Nippon Roche Research Center, 200 Kajiwara, Kamakura-Shi, Kanagawa 247, Japan. CHRIS L. GREER Department of Biological Chemistry, College of Medicine, D240, Med Sci I, University of California, Irvine, CA 92717, USA. ARNOLD HAMPEL Department of Biological Sciences, Northern Illinois University, De Kalb, Illinois 60115-2861 , USA. B. DAVID HAMES Department of Biochemistry and Molecular Biology, University of Let!ds, Leeds LS2 9JT. STEPHEN J. HIGGINS Department of Biochemistry and Molecular Biology, University of Let!ds, Leeds LS2 9JT. WALTER KELLER Abteilung Zellbiologie, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. TOM MANIA TIS Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. CRA IG L. PEEBLES Department of Biological Sciences, 365 Crawford Hall, University of Pittsburgh, Pittsburgh, P A 15260, USA. JEFF ROSS McArdle Laboratory for Cancer Research, University of Wisconsin··Madison , 1400 University Avenue, Madison , Wisconsin 53706, USA.

Contributors DAVID A . SHUB

Center for '\ltolecular Genetics, Depanmem of Biological Sciences 126, State University of New York at Albany, 1400 Washington Avenue, Albany NY 12222, USA . AARON J. SHATKlN

Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, Ne,-.. Jersey, NJ 08854-5638, USA. LARRY SIMPSON

Howard Hughes Medical Institute, Department of Biology, University of California, Los Angeles, 10833 Le Coate Avenue, Los Angeles, California, CA 90024-1662, USA. AGDA M. SIMPSON

Department of Biology, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California, CA 90024-1606, USA. BARBARA SOLLNER-WEBB

Department of Biological Chemistry, School of Medicine, The Johns Hopkins University, 725 Nonh Wolfe Street, Baltimore, MD 21205-2185, USA. ELMAR WAHLE

Abteilung Zellbiologie, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

XX

Abbreviations A Am

A260 A600 AdoHey AdoMet AMP ASV ATP bp BHI BHK BSA

c

eDNA Ci

CIP CMP c. p.m.

CPSF CPV

CTP D DAPI dATP ddATP DEAE DBAE

DEPC DHFR DMEM DMSO DNase d. p.m.

DTE DTT EBS EDTA

EGTA

adenine/ adenosine/ adenylate absorbance (259 nm) absorbance (260 nm) absorbance (600 nm) S-adenosylhomocysteine S-adenosylmethionine adenosine 5 1 -monophosphate avian sarcoma virus adenosine 5 1 -triphosphate base pair(s) brain heart infusion baby hamster kidney bovine serum albumin cytosine/ cytidine/ cytidylate complementary DNA curie calf intestinal alkaline phosphatase cytidine 5 1 -monophosphate counts pe1r minute cleavage and polyadenylation specificity factor cytoplasmic polyhedrosis virus cytidine 5 1 -triphosphate dihydrouridine 4 1 ,6 1 -diamidino-2-phenylindole deoxyadernosine 5' -triphosphate dideoxyadenosine 5 1 -triphosphate diethyl aminoethyl dihydroxyboryl aminoethyl diethyl pyrocarbonate dihydrofolate reductase Dulbecco's modified Eagle's medium dimethyl sulphoxide deoxyribonuclease disintegrations per minute dithioerythritol dithiothreit.ol exon binding sequence ethylenediamine-tetraacetic acid ethylene glycol-0 1 0 1 -bis(2-aminoethyi)-N,N,N 1 ,N 1 -tetraacetic acid

Abbreviations elF ETS

FPLC g

G

GMP gRNA

GTP h HGPRT

HIV HPLC 1BS ig

IPTG ITS kapp

kbp kcat

kDa kDNA Km kRNA

LDH rn 1A

m6A

m3G m7G min mRNA mRNP

nt

NADH NDK

NES NTP ori

PABP

PAGE

PBS PCR

PCV

PEG PEl

eukaryotic initiation factor external transcribed spacer fast-performance liquid chromatography gravit) guanine/ guanosine/ guanylate guanosine 5 1 -monophosphate guide RNA guanosine 5 1 -triphosphate hour(s) hypoxanthine-guanine phosphoribosyltransferase human immunodeficiency virus high-performance liquid chromatography intron binding sequence internal guide (sequence) isopropyi-{3-D-thiogalactoside internal transcribed spacer apparent equilibrium constant kilobase pair(s) turnover number kilodaltons kinetoplast DNA Michaelis constant kinetoplast RNA lactate dehydrogenase !-methyl adenine 6-methyl adenine 2,2, 7-trimethyl guanosine 7-methyl guanine minute(s) messenger RNA messenger ribonucleoprotein (particle) nucleotide(s) reduced nicotinamide dinucleotide nucleoside diphosphate kinase sodium acetate, EDTA, SDS buffer nucleoside 5 1 -triphosphate origin of replication poly(A)-binding protein polyacrylamide gel electrophoresis phosphate-buffered saline polymerase chain reaction packed cell volume polyethylene glycol polyethylenimine xxii

Abbre\·iations phosphoenolpyruvate pre-edited region inorganic phosphate pyru\'ate kinase PK phenylmethylsulphonyl fluoride PMSF packed nuclear volume PNV pans per million p.p.m. pounds per square inch p.s.i. polyvinyl alcohol PYA polyadenylic acid Poly(A) Poly(A)-RNA polyadenylated RNA polycytidylic acid Poly(C) ribonuclease RNase ribonucleoprotein (particle) RNP ribonucleoside 5 ' -triphosphate rNTP revolutions per minute r.p.m. ribosomal RNA rRNA Renografin, sucrose, EDTA buffer RSE Renografin, sucrose, Tris, EDTA buffer RSTE ribosomal salt wash RSW s svedberg SDS sodium dodecyl sulphate SDS-PAGE SDS-polyacrylarnide gel electrophoresis sec second(s) SER spliced exon reopening SET salt, EDTA, Tris buffer SJH splice junction hydrolysis snRNA small nuclear RNA snRNP small nuclear ribonucleoprotein (particle) sse standard saline citrate STE sucrose, Tris, EDTA buffer STM sucrose, Tris, MgC1 2 buffer SV40 simian virus 40 T ribothymidioe/ ribothymidylate TAE Tris-acetate-EDTA buffer TBE Tris-borate-EDTA buffer TCA trichloroacetic acid TE Tris-EDTA buffer TEAB triethylammoojum bicarbonate TEMED N,N,N ',N '-tetramethylenediamine TLC thin layer chromatography Tm melting temperature TMA tetramethylammonium chloride TMN Tris, MgCI 2, NaCI buffer

PEP PER P;

xxili

Abbreviations TMY tRNA TS TSE TUTase

u UDP UMP UTP

uv

v V;

v,

w X-gal it

tobacco mosaic virus transfer RNA Tris saline Tris, SDS, EDTA buffer terminal uridylyl transferase uracil!uridine/ uridylate uri dine 5' -diphosphate uridine 5 1 -mono phosphate uridine 5 1 -triphosphate ultraviolet volt(s) initial velocity resuspension volume watt(s) 5-bromo-4-chloro-3-indolyl-~-o-galactoside

pseudouridine

:xxiv

3' end-processing of m.RNA ELMAR WAHLE and WALTER KELLER

1 . Introduction: overview of 3 ' end-processing in

mammalian cells The 3 1 -end of a eukaryotic messenger RNA is not formed by precise termination of transcription. Rather, RNA polymerase II synthesizes a precursor RNA extending far beyond the 3 1 -end of the mature mRNA. The end of the mature RNA is then generated by post-transcriptional processing of the precursor RNA. Formation of the 3 1 -end is an essential step in mRNA synthesis; mutations in the RNA sequences that direct this process lead to a strongly reduced steady-state level of mRNA. In mammalian cells, almost all mRNAs receive their 3 1 -ends in a process consisting of two reactions: (a) The precursor RNA is first cleaved endonucleolytically at a particular site 3 1 (downstream) of the coding sequences. 1 (b) The 5 (upstream) fragment is then extended by the addition of approximately 200 adenylate residues (po1yadeny1ation), whereas the downstream fragment is degraded. The reactions depend on two sequences in the precursor RNA: the highly conserved sequence AAUAAA 10-30 nt upstream of the cleavage site and one or more sequence elements within approximately 50 nt downstream of this site. The downstream elements are not well defined; they may be either GU-rich or runs of U. In yeasts and plants, mRNAs are also polyadenylated. However, the sequence requirements and, possibly, mechanisms of 3 ' -end formation are different from those outlined above (for a recent review, see ref. 1). Cleavage and polyadenylation can be carried out in vitro in a nuclear extract from tissue culture cells (2). A complex sex of protein factors has been identified that catalyses both reactions in a tightly coupled fashion. At least two of these Proteins, and possibly more, are 'cleavage factors' involved only in the initial endonucleolytic scission; they are dispensable for polyadenylation. Two other factors are also required for the cleavage reaction but, in addition, carry out Polyadenylation. These are poly(A) polymerase, the enzyme that catalyses the Polymerization of the poly(A) tail from ATP, and a factor that binds to

1: 3' end-processing

of mRNA

AAUAAA and provides the polymerase with its specificity for RNAs containing this sequence. This latter factor has been described under different names; the authors will refer to it as cleavage and polyadenylation specificity factor (CPSF). In addition, a poly(A)-binding protein is involved in the elongation phase of poly(A) tail synthesis. For recent reviews, see refs I, 3. In mammalian cells, the only kno.... n exceptions to the mRNA processing pathway described above are the precursors to the major histone mRNAs. These undergo an endonucleolytic cleavage just downstream of a hairpin-loop structure. Poly{A) tails are not added. The reaction can be analysed in vitro (4). One of the factors required is the U7 snRNP. \1essenger RNA sequences involved in this form of 3' end-processing include a region which basepairs with the U7 RNA. The other factors involved in the reaction have nor been well characterized. For a review, see ref. 5. This chapter describes methods used to study endonucleolytic cleavage and polyadenylation of mRNA precursors in vitro. Procedures that are of basic importance and general applicability have been included. References will be given for procedures which are beyond the scope of this chapter or with which the authors have no personal experience. Section 2 deals with 3 ' end-processing in vitro in nuclear extracts from HeLa cells. Section 3 describes methods for the purification and assay of processing factors. Section 4 gives ad"ice on monitoring processing in vivo.

2. 3'-end cleavage and polyadenylation in

nuclear extracts 2.1 Introduction Processing of the 3 '-end of an RNA transcript may be studied in nuclear extracts with the use of appropriate RNA substrates synthesized in vitro. This section describes the preparation of nuclear extracts active in 3 ' end-processing and their use in studies of the formation of polyadenylation complexes, as well as of the cleavage and polyadenylation reactions themselves.

2.2 RNA substrates for 3' end-processing in vitro In vilro. RNA processing reactions are most easily analysed by the use of radiolabelled RNA substrates. These precursors are synthesized generally by 'run-ofr transcription of appropriate gene sequences cloned downstream from promoters for bacteriophage-encoded RNA polymerases (e.g. T3. T7, or SP6 polymerases). Protocols for the synthesis of RNA substrates and their purification by denaturing polyacrylamide gel electrophoresis are described in Volume I, Chapter I (Section 2). The following practical points should be observed when RNA substrates for m vitro processing are synthesized: 2

Elmar Wahle and Walter Keller (a} There is some suggestion that the 5' -cap structure of an RNA transcript plays a role in 3 ' end--processing, though this is still controversiaL The cap certainly stabilizes the RNA during the incubation in nuclear extracts. In general, capping of the RNA substrate is, therefore, desirable. (Volume I, Chapter I, Section 2.2 provides a protocol for the synthesis of capped RNA substrates.) (b) RNA substrates of different specific radioactivities are required for different assays (see Section 2.5). Make RNA of the desired specific radioactivity by adjusting the ratio of labelled to unJabelled UTP in the transcription reaction (see Volume I, Chapter I, Section 2.2). (c) In vitro processing reactions shouJd be carried out with known quantities of the RNA substrate. Calculate the concentration of radioactive RNA synthesized from the amount of radioactivity incorporated (count an aliquot of the RNA preparation in a scintillation counter), the specific radioactivity of the [ 32P] UTP used in the transcription reaction, and the number of UMP residues in the RNA. Further advice is given in Volume I, Chapter I, Section 2.3.4. (d) Store the RNA as an ethanol precipitate at - 20 °C. Brief vortexing of this should be sufficient to generate a homogeneous suspension so that the desired quantity of RNA for use in processing reactions can be withdrawn. If a homogeneous suspension cannot be generated for accurate and reproducible sampling, precipitate the RNA (see Protocol 5, steps 1-3), dissolve the pellet either in water or 5007o ethanol, and store it at - 20 oc. In these cases, lyophilize an aliquot of the RNA prior to use. (e) If the RNA is to be used as a 'precleaved' substrate for polyadenylation (see Section 2.5.4), a free hydroxyl group at the 3'-end is essential. Both chemical hydrolysis of RNA and degradation by the majority of nucleases leave a phosphate group at the 3 '-end which prevents polyadenylation. While RNA synthesized by the standard run-off procedure is generally a good substrate for polyadenylation, the authors have encountered phage T7 RNA polymerase from one supplier which generated RNA that could not be extended by poly(A) polymerase. The quality of the RNA 3 ' -ends can be checked by a preliminary nonspecific polyadenylation procedure. For this purpose, Mnh -dependent extension with unlabelled ATP followed by gel electrophoresis (see Section 2.5.5) shouJd be used for radiolabelled RNA. Ext.ension with radiolabelled A TP followed by TCA precipitation (see Protocol 8) should be used for unlabelle-d RNA substrates. Many studies of 3 end-processing in vitro have used two particular polyadenylation sites: the site in SV40 late mRNA and the L3 site of adenovirus 2. 3

1: 3' end-processing of mRNA If a polyadenylation site is to be used that has not been characterized before, the following points should be noted:

(a) Use a genomic fragment rather than a eDNA fragment to generate the RNA substrate since the essential downstream elements will not be present in the latter. Design the run-off transcription so that at least 50 nt downstream of the polyadenylation site are included in the precursor RNA. (b) Some genes contain additional 3' end-processing signals upstream of the

AAUAAA sequence. Therefore, include in the RNA substrate about 150 nt upstream of this sequence. (c) To test whether any processing event observed is genuine, use an RNA precursor with a point mutation in the AAUAAA sequence (e.g. AAGAAA) as a control.

2.3 Preparation of nuclear extracts for 3' end-processing Because the substrate for most RNA processing assays is a small quantity of radiolabelled RNA, RNases pose a major problem. In practice, the high abundance of RNases in extracts of many tissues limits the choice of starting material for extract preparation to cultured cell lines. Nuclear extracts from HeLa cells are the standard material for the analysis of RNA processing in vitro because they are low in endogenous RNases and can be readily prepared. The following procedure (Protocol 1) uses freshly-harvested HeLa cells for the preparation of nuclear extract, but it also works with cells that have been frozen. However, fresh cells swell better in the hypotonic buffer and thus can be more easily lysed. The use of a Dounce homogenizer for cell lysis requires a certain minimal volume (approximately 2 ml for a small homogenizer). If small amounts of cells are to be processed, they can be taken up in a syringe and forced repeatedly through a 25-gauge needle (7). Alternatively, cells are not broken mechanically but lysed by the addition of lysolecithin (8). Usually, the nuclear extract is dialysed before use since the salt concentration limits the amount of non-dialysed extract that can be added to a processing reaction. However, the non-dialysed extract seems to be more active in the cleavage assay (see Protocol 6 and Section 2.5.3). Note that the buffers used to prepare the nuclear extract (see Protocol/) contain 1.5 mM Mg2 +, whereas dialysis buffer (buffer D; see Protocol 1) does not. The amount of Mg2 • added to in vitro reactions may thus have to be adjusted. Although Protocol I was developed for use with HeLa cells, extractS from other cell lines can generally be made by the same procedure. However, the protocol cannot be applied to the preparation of nuclear extract from solid tissue (e.f. calf thymus, see Section 3.2.3). 4

Elmar \Vahle and \\'alter Keller Protocol 1. Preparation of Hela cell nuclear extract for 3 ' end-processinga Equipment and reagents • Oounce homogen1zer lcapac1ty 120 ml for 151itres of Hela ce ll culture) with a type S pestle

• H1gh-salt buffer C (buffer C contaimng 1.2 M NaCI)

• Phase-contrast m1cros<:ope • 1.0 M OTT 1n 10 mM sod1um acetate, pH 4 .8. store at - 20 .,C e 0.2 M PMSF 1n absolute ethanol or Isopropanol, store at -- 20 •c • Buffer A• (10 mM Hepes- KOH , pH 7.9<, 1.5mM MgCI 2, 10mM KCI, 0. 5 mM OTT" I e Buffer c• (25% glycerol, 20 mM HepesKOH . pH 7 9'. 1. 5 mM MgCI2 , 0. 2 mM EOTA, 20 mM NaCI, 0.5 mM OTT", 0.5 mM PMSF• )

e Buffer 0 ° 120 mM Hepes-KOH, pH 7.g. , 20% glycerol, 100 mM KCI, 0 2 mM EOTA 0 5 mM OTT" I • Phosphate-buffered sal,ne IPBSlc 140 g NaCI, 1 g KCI, 5.75 g Na2 HP04 , 1 g KH 2P04 , H2 0 to 5 litresl. the pH should be 7.2 • Hela cells grown in suspension culture to 4-5 x 10s cellslml in standard tissue culture medium !e.g. Joklik's minimal essential medium) plus 5% newborn calf serum

Method 1 . Harvest the Hela suspension cells by centrifugation at 1 300 g.,... for 10 m1n at room tHmperature. 2. Resuspend the cells in a small volume of PBS and transfer the suspension to graduated Falcon tubes. Centrifuge as in step 1. 3. Decant the supernatant and determ1ne the packed cell volume (PCV). Resuspend the cedis in PBS to 5 x PCV and centrifuge as in step 1.

4 . Carry out this and all subsequent steps on ice. Resuspend the cells in icecold buffer A to the cells swell.

~~

x PCV . Leave the suspension on ice for 10 min while

5. Homogenize the c:ells by 10 strokes in the Dounce homogemzer on 1ce, checking for complete cell lysis by phase-contrast microscopy'. 6 . Centrifuge the homogenate in graduated Falcon tubes for 1 5 m1n at 2000 x gmax. 7 . Measure the total volume of the homogenate. Carefully take off the supernatant and measure 1ts volume. Calculate the difference between total volume and supernatant volume. This difference is the packed nuclear volume (PNV). 8 . Resuspend the nuclei in ice-cold buffer C to 0 . 5 x PNV. Slowly add highsalt buffer C (0. 5 x PNV) dropwise and with swirling". 9 . Homogemze the nuclei by 10 strokes in the Dounce homogenizer on ice. Transfer the hom.:>genate to a beaker in an ice bath and sttr 1t gently for 30 min on a mag netic st1rrer. 10 . Centrifuge the ho,mogenate for 30min at 16000 x gmax·

Continued

5

1: 3' end-processing o.f mRl\'A Protocol 1. Continued 11. Remove the supernatant (nuclear extract) D1alyse (optional) the extract for 5 h against 2 litres of buHer D

12 . Freeze the nuclear extract 10 suttable aliquots 1n liquid nitrogen and store them at -70 °C. ' A mod1flcat1on of the ong1nal protocol of Dignam et a/. (6) developed by Robin Reed (Harvard Medical School! and communicated to the authors by Ryszard Kole (University of North Carolina). • All buffers can be stored for several weeks in a refrigerator. • Adjust a 1 .0 M stock solution of this compound at room temperature to the pH indicated. Use this stock to make up the buffer without further adjustment of the pH. d Add OTT just prior to use from the 1.0 M stock solution. • PMSF must be added dropwise with vigorous st1rr1ng just prior to use. ' The number of strokes suggested is meant as a guide. Monitonng of cell lysis by microscopy is essential. • The suspension should become only slightly v1scous. If high·salt buffer is added too fast. DNA will be released. makmg the extract too viscous for homogenization and centnfugat1on

2.4 Formation of polyadenylation complexes 2.4.1 Introduction The endonucleolytic cleavage at the sile of future poly(A) addition only occurs after the substrate RNA has been assembled into a large complex that contains some or all the trans-acting processing factors. The composition of this complex is not known precisely since its formation has so far been analysed only in crude extracts. It is, however, quite clear that binding of CPSF to the AAUAAA sequence is the key event for complex formation (9). One of the cleavage factors may interact with a downstream sequence element and stabilize the CPSF-AAUAAA interaction (10). It is not known whether any of the other factors is stably associated \\ith the processing complex. A variety of experimental approaches can be made to investigate polyadenylation complexes:

(a) The formation of processing complexes may be detected by either glycerol gradient sedimentation {II) or, more conveniently, non-denaturing gel electrophoresis (see Section 2.4.2). (b) RNA can be extracted from the complexes. Analysis of this RNA by denaturing gel electrophoresis will reveal whether a complex contains substrates, intermediates, o r products of the reaction. (c) The region of an RNA substrate associated with processing factors can be investigated by nuclease digestion or chemical probing techniques (see Sections 2.4.3 and 2.4.4). 6

Elmar \Vahle and Walter Keller (d) Polyadenylation complexes separated by nati\e gel electrophoresis may be anal}sed with respect to their protein content by subsequent SDS polyacrylamide gel electrophoresis. UV cross-linking to the substrate RNA can identify those proteins that are in close contact with the R1 A (9). (e) The presence or absence of particular antigens or nucleic acids (e.g. snRNAs) in the polyadenylation complexes can be tested with blotting techniques (12).

2.4.2 Basic method Protocol2 describes the formation of processing (or polyadenylation) complexes using the nuclear extract prepared in Protocol 1. Protocol 2 forms the basis for many of the subsequent procedures. With slight modifications, it can also be used to analyse the binding of purified CPSF to AAUAAA-containing RNAs (see Section 3.2.5) or other RNA-protein interactions. Tbe polyadenylation complexes generated in Protocol 2 are visualized by nondenaturing (native) polyacrylamide gel electrophoresis. Complex formation requires ATP and so creatine phosphate is included in the reaction to replenish the ATP pool that is depleted b} ATPases in the nuclear extract. The extract contains adequate amounts of creatine kinase. Polyvinyl alcohol (PVA) is included in the reaction because it stimulates many macromolecular interactions, probably by increasing the effective concentrations of the reacting molecules ('excluded volume effect'). After the polyadenylation complexes have formed, heparin is added. Heparin is an anionic polymer that binds many nucleic acid-binding proteins and here serves to remove non-specific RNA-binding proteins from the precursor RNA. Protocol 2. Formation of polyadenylation complexes and their analysis by electrophoresis in native gels Equipment and reagents • Equipment for polyacrylamide gel electrophoresis

e 10mM MgCI7

• Dialysed Hela cell nuclear extract (see Prococo/ 11

e 12 5 % polyv1nyl alcohol IPVA. low molecular we•ght cold water soluble; S•gma)

•

e 2. 5 mg lml tRNA

32 P-Iabelled precursor RNA (approximately 500-1000 c.p.m fmoll. synthesrzed and capped tn vtrro (see Volume I. Chapter 1, Secuon 2.2)

• Hepaun 125 mg ml Sigma. grade I) • Loadmg buffer (10% glycerol 0.05% xylene cyanol, 0 .05% bromophenol blue)

• 0. 2 M OTT 1n 10 mM sodium acetate. pH 4 .8 . store at - 20 ° C

• Native gel electrophoreSIS buffer (25 mM Tris base, 25 mM borrc ac1d, 1 mM EDTA)•

• 10 mM A TP Dissolve A TP 1n water at approximately 0 1 M . Neutralize With ammoma, check1ng the pH with Indicator paper Determine the exact concentration by measuring the A 259 1~ 259 = 15 400). Store this stock solution at - 20 ° C. Dilute aliquots to 10 mM as required and store them also at - 20 ° C

• Acrylamide stock solution (20% acrylamide, 0 .25% bisacrylam1de in native gel electrophoreSIS buffer) • 10% ammonium persulphate (freshly made)

• 0 . 2 M creatine phosphate

eTEMED

7

Continued

1: 3' end-processing

of mRNA

Protocol 2 . Continued Method A. Preparation of the non-denaturing polyacrylamide gel

1. Clean glass electrophoresis plates, spacers, and, most of all, the comb thoroughly. 2. Assemble the glass plates (20 em long) with 1 mm thick spacers.

J . Make up a 4% polyacrylamide solution in nattve gel electrophoresis buffer tn a sufficient volume for the gel apparatus being used. For each 100ml mix: • acrylamide stock solution enative gel electrophoresis buffer

20ml 80ml

0.1 ml e TEMED 0.75ml • 1 0% ammonium persulphate 4. Pour the acrylamide solution between the glass plates, insert a well-forming comb, and leave the g•el to polymerize.

B. Preparation of the polyadenylation complexes 1. Prepare a reaction mtxture suffictent to enable 12 x 25 1-11 timed samples to be taken. To do this, dispense 120 fmol of RNA precursor (equivalent to 10 ng of an RNA 2Ei0 nt long) tnto a mtcrocentnfuge tube. Calculate the volume of RNA solution to be used from its concentration determined as described tn Section 2. 2. 2 . If the RNA has been stored as an ethanol precipitate, centrifuge it for 15 min in a microcentrifuge at 4 °C. Remove the supernatant as completely as possible since it contains the urea from the gel that was used to purify the RNA (see Volume I. Chapter 1, Section 2.3.3). Add 1 ml of 70% ethanol to the RNA pellet. centnfuge for 5 mtn, discard the supernatant and dry the pellet. Use a hand-held Ge1ger counter to control the recovery of radtoacttve RNA tn this and all subsequent steps. Proceed to step 4. 3 . If the RNA has already been precipitated and stored tn water or 50% ethanol, lyophtltze the appropnate quanttty 4. To dissolve the RNA pE!IIet, add the followtng reagents in the order gtven . • 2.5mg ml tRNA • H2 0 .o.2M

5

on

121-11 61-11 31-11

•10mM ATP

241JI

• 0.2 M creatine phosphate

30 1-11

• 10 mM MgCI 2

1 5 1-11

Vortex to mix and dissolve the RNA. Place the tube on ice and add 60 IJI of 1 2.5% PVA and 1 50 1-11 of nuclear extract • Mix gently by ptpetttng

6 . Wtthdraw a 251-11 aliquot into a fresh microcentrifuge tube and freeze it on dry ice.

Continued 8

Elmar \Vahle and Walter Keller Protocol 2. Contmued

7 . Transfer the rest of the reaction mixture to a 30 °C water bath. Take additional 25 ~I aliquots after 1, 2, 5, 10. 20, 30, 40, 50, 60, and 120 min. Freeze each on dry ice.

8 . At the end of the incubation, take all the tubes from the dry ice. Add 5 ~I of 25 mg/ml heparin to each and leave them for 10 min at room temperature.

C. Electrophoresis of polyadenylation complexes 1. Remove the comb from the non-denaturing polyacrylamide gel, assemble the apparatus and rinse the wells w1th native gel electrophoreSIS buffer.

2 . Prerun the gel for at least 1 h at 250 V . 3 . Load aliquots of all the samples into the wells of the gel. Use aliquots compatible with the size of the wells (5-30 ~1). 4 . Mix an aliquot of substrate RNA, that has not been incubated with proteins, with loading buffer. Load this sample into an empty lane of the gel•. 5. Run the gel at 2 50 V. The running time depends on the length of the precursor RNA; for an RNA of 250 nt, run the xylene cyanol marker dye in the lane containing free substrate RNA to the bottom of the gel 6 . Remove the gel from the electrophoresiS apparatus and separate the glass plates. 7 . With the gel on one of the plates, wrap 1t 10 plast1c cling film (e.g. Saran wrap). Place an X-ray film 1n contact with the gel and use a marking pen to mark the orientation of the gel on the film . 8. Expose the film at - 70 °C for an appropriate time and then develop 1t according to the manufacturers instructions. • Note that this buffer IS different from the TBE buffer normally used for gel electro· phoresis. ~ Less nuclear extract may be used. If so. add buffer D Isee Protocol 1) so that the combined volume of nuclear extract and buffer D is one half of the total reaction volume. ' loadmg buffer IS added only to the free substrate ANA but not to the aliquots from the reaction. These con tam suff1c1ent glycerol for loadmg .

The experiment shown in Figure /A demonstrates the time course of polyadenylation complex formation. The fastest migrating complex (complex H) represents the non-specific binding of proteins to the RNA. [ts formation does not require any of the polyadenylation signals. A second complex (S), the specific PQiyadenylauon complex, forms only ""ith RNAs that contain aJlthe sequences necessary for 3 ' end-processing. In the presence of ATP, this complex forms rapidly even at 0 °C. RNA that was at first bound by non-specific components (complex H) is also incorporated into complex S. Later, a third complex (P} appears that contains the cleaved and polyadenylated RNA product (see Section 2.5.2}. 9

1: 3' end-processing

of mRNA

A ..... X

z

I 0 I

2 5 '0 20 )() <0 SO 60 IJO rT\11'1

....... .. ·-

• ... - - "' ' ,,

.......... '

. -ong1n

-s -P

lH

•

-pre

B

II :polyiAl -------~ -pre Figure 1 T1me course of the formatiOn of polyadenylatton complexes and polyadenylated RNA Polyadenylauon complexes were formed accordtng to Protocol 2 on an RNA (246 ntl conte1mng the adenovtrus l3 polyadenylation Site. fA) Complexes were analysed on a non denaturong 4% polyacrylam•de gel. For an explanat•on of the complexes formed, see the text. Precursor RNA IS tnd•cated by 'pre'. A control lane w1th RNA in the absence of nuclear extract is labelled NXT'. IB) RNA was punf1ed from al,quots of the same samples as in lA I and analysed on a 6% denaturing ge Reproduced from ref 13.

In the procedure described in Protocoll, RNA can be purified from a second aliquot of each sample and analysed on a denaturing gel so that a time course of the processing reaction itself is obtained (for a detailed discussion, set. Secuon 2.5.2 and Protocol 6). Although complex formation is very rapid polyadenylated product appears only after a significant lag period (compare Figures JA and JB). The RNA present in each of the various complexes can be analysed as described (ref. 13; see also Section 2.4.3, Protocol 3). 2.4 .3 Analysis of poly adenylation complexes by RNase protection RNA sequences involved in the formation of the polyadenylauon complex are protected by the binding of processing factors agamst digest1on wnh RNase Tl . 10

Elmar \\'ahle and Walter Keller This nuclease cleaves R A on the 3 ' -side of GMP residues, producing a pattern of oligonucleotides that~:; character~tic for every RNA and that can be predicted from its sequence. In Protocol 3, the polyadenylation complexes are treated with a low concentration of RNas•e Tl and then separated on a non-denaturing gel. After the polyadenylation complexes have been isolated from the gel, they are deproteinized and their protected RNA fragments are further analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. In the final stage of the procedure, each of these fragments is isolated from the denaturing gel and subjected to a second RNase Tl digestion and denaturing gel electrophoresis. RN.ase Tl cleavage sites that were protected during the frrst digestion will be a•ccessible during the second digestion. A comparison of the oligonucleotides generated from the protected fragments during the second digestion to the ,pattern of oligonucleotides generated from the naked substrate RNA will identify those sites that were protected during the first digestion and thus the regions of the RNA involved in polyadenylation complex formation . The polyadenylation complex is generated as described in Protocol 2. However , for the efficient detection of RNA fragments after the various RNase digestions, the RNA sulbstrate must have a higher specific radioactivity.

Protocol 3. Analysis of polyadenylation complex formation by

RNase T1 protection Equipment and reag'ents • Non-denaturing polyacrylamide gel and electrophoreSIS equ1pment (see Protocol 21 • Two denatunng polyacrylamide (sequencing) gels•, one W ith 12% acrylamide and the other w1th 20%, TBE runnmg buffer, and formam1de loading buffer (see Volume I, Chapter 1. Protoco/81 • RNase T1 !Calbiochem). Make a stock solution (50 unltS/)Ji) in 10 mM Tris-HCI , pH 7.5. 1 mM EDTA and dilute this in the same buffer as required • Proteinase K I 10 mg 'rnl ; Boehringer Mannheiml

•

32 P·Iabelled RNA substrate 120 000 c .p.m 'fmol) 0 , d1alysed Hela cell nuclear extract, and reagents for generating the polyadenvlauon complex (see Protocol 2 1

•TBE buffer (25 mM Tris base, 25 mM bone acid 1 mM EDTAl containmg 0. 1% SDS •TE buffer (10 mM Tris - HCI, pH 7.5, 1 mM EDTAI containing 2.5 mg/ml tRNA and 1 unit/)Ji RNase T1 • Phenol:chioroform I 1:1 l

e 32 P·Iabelled DNA electrophoresiS SIZe

• RNA elution buffer (0. i7 5 M ammomum acetate, 10 mM magnesium acetate, 1% (v/v) phenol, 0 1% SDS. 25 11g ml tRNA)

markers covering the range between 20 and 100m (e.g. pBR322 d1gested w1th Hpall and end-labelled with [ ">-32P 1A TP and polynucleoude kinase; see Volume I, Chapter 2, Section 3 .2 2)

Continued 11

1. 3 ' end-processing of miL\iA Protocol 3 . Continued

Method 1. Set up a 251-11 polyadenylation reaction as 1n Protocol 28. To do this, dispense approximately 107 c.p.m. of labelled precursor RNA into a microcentrifuge tube.

2. Depending on the way the RNA has been stored, centrifuge or lyophilize the RNA 1n a microcentrifuge tube as described in Protocol 28, steps 2 or 3 . In this and all subsequent steps, control the recovery of RNA with a hand-held Ge1ger counter.

3. To the RNA sample (step 2) add: e 2. 5 mg/ml tRNA

1 1-11

e 10mMATP • 0. 2 M creatine phcJsphate e 70mM on e 10 mM MgCI 2

21-11 2 .51JI 0.751-11

1.25!-11 4 . Vortex to dissolve the RNA . Place the tube on ice and add 51JI of 12.5% PVA and 12. 5 IJI He La cell nuclear extract. 5 . Mix gently by pipetting and incubate for 30 min at 30 ° C. 6 . Add 0 . 1 unit of RNa se T1 and incubate the m1xture for 10 min at room temperature. 7 . Add 25 mgl ml heparin to 0.2 mg/ml final concentration. 8. Incubate for 10 min at room temperature then load an aliquot (at least 5 IJI) or all of the sample onto the non-denaturing polyacrylamide gel. Separate the complex by electrophoresis as 1n Protocol 2C. 9 . After electrophoresis , wrap the gel in cling film. Place an X-ray film on the gel and use a fibre-tip pen to apply several guide marks extending from the X-ray film to the cling film . Expose and develop the film . 10. Place the gel on top of the autoradiograph and use the marks to align the film. Using the autoradiographic image as a guide, and using a clean scalpel, cut out a gel pcece containing the specific polyadenylation complex". 11 . Soak the gel piece in TBE buffer contaimng 0 1% SDS for 10 min at room temperature, then incubate the gel slice for 30 mm at 30 ° C in TBE buffer containing 0 . 1% SIDS and 0 .5 mg/ml proteinase K. 12. Using clean forceps , place the gel piece in a well of the 12% denaturing polyacrylamide gel. 13. Mix radioactive DNA size markers with formamide loading buffer. Heat the solution for 3 rnin to 95 °C , chill it on 1ce, and load it into one of the wells of the denaturing gel. 14. Run the gel unt1l the bromophenol blue marker has reached the bottom of the gel. 15. Autoradiograph the gel and cut out gel pieces containing the major protected RNA fra•gment(s) as in step 10.

Continued 12

Elmar Wahle and Walter Keller

Protocol 3. Continued 16. Incubate each gel fragment separately overn1ght at 37 °C

1n

400 IJI of

RNA elut1on buffer.

17. Remove the buffer from the gel fragments into a fresh m1crocentnfuge tube.

18. Extract the eluted RNA by vortexing w1th an equal vol. of phenol:chloroform. 19. Centrifuge in a microcentrifuge for 30 sec to separate the phases. 20. Remove the upper (aqueous) phase into a fresh microcentrifuge tube. Add 2.5 vol. of ethanol, mix, and centrifuge for 15 min in a microcentrifuge. 21 . D1scard the supernatant. Add 1 ml of 70% ethanol to the tube and centrifuge 1t again for 5 min. Discard the supernatant and dry the pellet under vacuum. 22. Dissolve the RNA oligonucleotide in 41JI of TE buffer containing 2. 5 mg/ml tRNA and 1 unit/IJI of RNase T1. Similarly set up a sample of the original RNA substrate for RNase T1 digestion as a control. 23. Digest the samples for 30 min at 30 °C. 24. Lyophilize each sample and then dissolve them in formamide loading buffer. Boil each for 3 min and chill on 1ce.

25. Load the samples onto the 20% denatunng polyacrylamide gel. Run the gel until the bromophenol blue marker

IS

10 em from the bottom

26 . Autoradiograph the gel. • The 12% gel should be of the same thickness as the native gel (1 mml so that gel fragments from the nat1ve gel can be placed on top of the denatuung gel. The 20% gel should be a normal (thin) sequencing gel. b Make RNA of the requ~red specific activity by using the appropriate spec1fic activity of [a·32P] UTP in the transcription reaction. Obviously, the intensity of the autoradiographic bands in the gels (steps 9, 15. and 26) depends on the number of UMP residues in each RNase T1 fragment. Fragments containing no UMP res1dues will not be visible . It may thus be more convement to label the RNA with [a-32P]GTP. Smce every fragment contams only a single GMP residue at its 3 '-end, th1s will result in uniform labelling intensity of all fragments. • The RNase T1 digestion may change the pOSition of the spec1f1c complexes u1 the gel. Non-specific complexes may disappear.

The RNase Tl digestion pattern of an RNA can be predicted from its sequence and most of the larger digestion products can be identified unambiguously by their size. Thus, the protected RNA fragment obtained from the polyadenylation complex can also be identified by the pattern of oligonucleotides generated from it in the second RNase Tl digestion. If a fragment obtained in this second digestion cannot be identified by its size alone, an additional digestion, e.g. ''ith RNase T2, may be used to determine its nucleotide composition (14). The size of the protected fragment before the second digestion gives an estimate of the total size of the binding site. This is significant because the distribution of GMP residues in the substrate RNA may not allow the identification of every oligonucleotide produced in the second digestion. 13

1:

3' end-processmg of mRNA

A

c

8

Tl

-

.

u

+

L

N U L 21--

00-

· --21

90-

s-

1~::: ~

67-- 7a-

H-

~~---

- -!5

11-9--· ··-1()

108- -

76- • • --6

pre - e

52

3

2

1 2 3

Ftgure 2 . Protection of precursor RNA from RNase T1 digestion by polyadenylation complex formation PolyadenylatiC)n complexes were formed on the same RNA as 1n Figure 1 120 mtn incubattonl and treated with RNase T1 (see Protocol 31. !AI Two such complexes, U (upper) and L llower), lane 3, were separated on a non -denatunng 4 % polyacrylamide gel after RNase T1 digest1on ( + ). The reason for the appearance of two specific complexes IS unknown. Control complexes IIane 2) were not incubated with RNase T1 1- ); 'pre' Indicates precursor not exposed to nuclear extract. 'H' (lane 1I represents a non-specific complex (see Figure 1) . (B) RNA oligonucleotides were purified from complexes U and L and artalysed on a denaturing 12% polyacrylamide gel (CI The 67 nt oligonucleottde from U and L was eluted from the gel, dtgested with RNase T 1 and analysed on a denaturing 20% polyacrylamide gel. The four fragments visible from U and L anse from both sides of the polyadenylation site N IIane 1 I shows RNase T1 digestion products from precursor RNA . The numbers indtcate s•zes (nt) of RNase T1 fragments or DNA s1ze markers. Reproduced from ref 13.

An estimation of the total binding site size from the sum of these secondary oligonucleotides may, therefore, not be possible. Figure 2 shows an example of RNase Tl protection. In the case of the adenovirus L3 polyadenylation site used in thls experiment, a fragment of 67 nt, containing the AAUAAA sequence as well as downstream sequences, is protected (Figure 28). A second RNase Tl digestion after purification of the protected 67 nt oligonucleotide generates several RNA fragments of between 6-21 nt , which lie on both sides of the polyadenylation site (Figure 2C). (The nucleotide sequence of the substrate RNA used in this experiment and its pattern of RNase T l digestions products can be found in ref. 13.) 14

J::Imar \\'ahle and Walter Keiler 2.4.4 Analysis of polyadenylation complex formation by

modification-·interference RNase protection yields only information about the segment of RNA that is stably bound by processing factors. In contrast, modification-interference analyses indicate which particular nucleotides are essential for the formation of processing complexes (15). ln this procedure, end-labelled substrate RNA is either modified chemtcally at purine bases \\ ith diethylpyrocarbonate or pyrimidine bases are removed with hydrazine. As in nucleotide sequencing by the chemical method. the extent of modification must be very low, so that less than one bas'e ts modtfied per RNA molecule. The modified RNA is used to form processing complexes. After non-denatunng gel electrophoresis, complexed RNA is recovered from the gel. Both thts RNA a pd substrate RNA that has not been incubated with protein are then cleaved at the sites of chemical modification and the cleavage products are separated on a sequencing (denaturing) gel. The non-complexed RNA provides gel tracks containing purine-specific or pyrimidine-specific cleavage ladders with which the RNA from the polyadenylation complex can be compared. If the modification of any panicu.lar nucleotide has interfered with complex formation, the band corresponding to this nucleotide will be missing in the ladders generated from the RNA sample isolated from the complex. The same procedure can be used to imestigate the binding of purified processing factors, such as CPSF to AAUAAA-containing RNAs. It can also be applied to study the formation of any other RNA-protein complexes. The method is not even limited to experiments in which a complex formed between proteins and the modified RNA is separated by native gel electrophoresis. Other methods (e.g. denaturing gel electrophoresis) can also be used to isolate the RNA that is accepted as a substrate in the reaction of interest and to separate it from the RNA that is excluded. The procedure for modificauon-interference (see Pr01ocol 5) uses an RNA precursor labelled at its 3 -end by a ligation procedure (see Protocol 4).

Protocol 4. 3' end-labelling of RNA substrates by ligation Equipment and reagents • Denaturing 6 % polyacrylamide gel, electrophoresis equipment. TBE butler. and formamide loading buffer !Volume t, Chapter 1. Protocol 8) • Unlabelled capped substrate RNA synthestzed as described tn Volume I Chapter 1, Protocol 4

• 15'-32P)pCp

!New Eng land Nuclear or Amersham· 3000 Cilmrnoll

e T4 RNA ligase (Pharmacia) e 10M

on (see Protocol

1)

e 10 x RNA ligase buffer (0 5 M Tns- HCI pH 7 .9. 0.15 M MgCI2 • 33 mM on•) • 0.25 mM ATP diluted from a stock solutton (see Protocol 2) • DMSO (Sigma!

• o 1 mg ml BSA

(nuclease free)

Continued

15

1: 3' end-processing of mRNA

Protocol 4. Continued

Method 1. In a microcentrifuge tube, mix 21-11 each of 0.25 mM ATP, 0.1 mg/ml BSA, 10xRNA ligase buffer, and DMSO plus 101-11 of [5'-32P]pCp. 2. Add 5-10 pmol of substrate RNA (equivalent to 80-160 ng of RNA 50 nt in length) in a volume of up to 2 1-11. 3. Cool the mixture to 4 °C and add 10-20 units of RNA ligase. 4 . Incubate at 4 °C for at least 10 h.

5. Purify full-length labelled RNA by denaturing polyacrylamide gel electrophoresis (see Volume I, Chapter 1, Protocol 8}. • Add DTI just before use from the 1.0 M stock.

Protocol 5 . Analysis of polyadenylation complex formation by modification-interference

Equipment and reagents • Non-denaturing polyacrylamide gel and electrophoresis buffer (see Protocol 2 ). 12% denaturing polyacrylamide gel (sequencing gel), TBE buffer. and formamide loading buffer (Volume I, Chapter 1, Protocol 8) together with the appropriate electrophoresis equipment • Electroblotting apparatus for wet blotting (e.g. Bio-Rad or Hoefer)

• 0. 3 M sodium acetate, adjusted to pH 3.8 with acetic acid • Diethylpyrocarbonate (DEPCI (Sigma)• • Anhydrous hydrazine !Eastman- Kodak)0 containing 0 . 5 M NaCI, prepare th1s solution by dissolving solid NaCI in hydraz1ne • 1.0% SDS

e DEAE transfer membrane (Schleicher and Schuell, NA45)

• 1.0 M aniline in 0 .3 M sodium acetate. pH 3.8, use redistilled an1line<

• Hela cell nuclear extract and reagents for generating polyadenylation complexes (see Protocol 2)

• Phenol. chloroform (1 :1 I

• 3 ' end-labelled substrate RNA (see Protoco/ 4 )

e o . 5 M NaOH

• Butan- 1-ol

e 10 mM EDTA, pH 7.6 • TBE buffer containmg 0 1% SDS (see Protoco/3 1

e 2. 5 mg/ml tRNA • 7 . 5 M ammonium acetate • 1.0 M sodium acetate, adjusted to pH 4 . 5 w1th acetic acid • 50 mM sodium acetate, pH 4 . 5, 1 mM EDTA, use the 1 M sodium acetate stock to make th1s solution without adjusting the pH

• NA45 elution buffer (55% formamide". 1.8 M sodium acetate, 2 mM EDTA, 0 .2% SDS). use solid sod1um acetate, not a solution whose pH has been adjusted • Urea loading buffer (8.0 M urea, 20 mM Tns-HCI, pH 7.9, 1 mM EDTA. 0 .05% xylene cyanol, 0 .05% bromophenol blue)

Continued

16

Elmor Wahle and Wolter Keller Protocol 5 . Continued Preparation of RNA substrates

1. Use precursor RNA as the substrate, synthesized without radioactive label as described in Volume I, Chapter 1, Section 2.2, and 3' end-labelled as Protocol 4. Mix two separate aliquots of this RNA (approximately 106 c.p.m. each) with 12.51Jg t RNA as carrier. Add 0.1 vol. of 7.5M ammonium acetate and 2. 5 vol. of absolute ethanol. Vortex and chill the mixture on ice for a few minutes. 2 . Centrifuge the mixture for 15 min in a microcentrifuge. Discard the supernatant. In this and all subsequent steps, control the recovery of RNA with a hand-held Geiger counter. 3 . Add 1 ml of 70% ethanol to the pellet. Centrifuge for 5 min in a microcentrifuge. Discard the supernatant and dry the pellet under vacuum . Purine modification 4 . Dissolve one RNA sample in 200 IJI of 50 mM sodium acetate, pH 4 . 5, 1 mM EDTA.

5 . Add 31JI of fresh DEPC and incubate the RNA for 5 min at 90 °C with the lid of the tube open. 6 . Add 751JI of 1 M sodium acetate, pH 4.5, and 900 IJI of ethanol. Collect the RNA precipitate as in steps 1 and 2. 7 . Dissolve the RNA pellet in 200 IJI of 0.3 M sodium acetate, pH 3.8. Precipitate the RNA by adding 600 IJI of ethanol. Collect the RNA precipitate as in steps 1 and 2. 8. Dissolve the RNA in 10 IJI of water. Store it at - 20 °C. Pyrimidine modification

9 . Dissolve the other RNA sample (steps 1-3) in 20 IJI of anhydrous hydrazine containing 0. 5 M NaCI and incubate it for 70 min at 4 °C. 10. Add 200 IJI of 0.3 M sodium acetate, pH 3 .8 , and 900 IJI ethanol, then collect the RNA precipitate as in steps 1 and 2. 11 . Dissolve and reprecipitate the RNA pellet as in step 7. 12. Dissolve the RNA in 101JI of water and store it at -20 °C . Complex formation and electrophoresis 13. Set aside one-tenth of each sample of modified RNA (steps 8 and 12) as control samples tor later aniline cleavage and electrophoresis steps. Use the rest of each sample separately for complex formation as described in Protocol 28 but use a reaction volume of 125 IJI. 14. Load each sample on the non-denaturing gel using several adjacent lanes to accommodate the entire sample. 15. Run the gel as in Protocol 2C .

16. After electrophoresis, transfer the RNAe onto an NA45 DEAE membrane by electroblotting in a wet blotting cell. To do th1s, cut out one piece of NA45 membrane slightly larger than the gel and soak it for 10 min in 10mM EDTA. pH 7.6, at room temperature.

Continued 17

1: 3' end-process ing Protocol 5 .

of mRI\'A

Contmued

17. Transfer the membrane mto 0 5 M NaOH and let It soak for 5 min. 18. Wash the membrane in 5 changes of water (2 mm each). 19. Cut out four pieces of Whatman 3MM paper of the same size as the NA45 membrane and wet them tn TBE buffer contatning 0 . 1% SDS . 20 . Place the NA45 membrane onto a stack of two pieces of 3MM paper. Place the gel directly onto the DEAE membrane and finally two sheets of 3MM paper on top. Be careful not to trap air bubbles. 21 . Assemble thts sandwich in the blotting cell according to the manufacturer' s instructions as for Western blotting. For transfer. use TBE buffer containing 0 .1% SDS. Blot for 6 h in a cold room at 50 V . 22. After the transfer is complete. wrap the DEAE membrane 10 cling film to prevent it drying and expose it to X-ray film ustng guide marks to align the membrane on the film (see Protocol 3, step 9). 23. Cut out strtps from t he membrane corresponding to the specific polyadenylation complex. 24. Elute the RNA from the DEAE membrane by soaking the membrane strips in NA45 elution buffer for 1 5 min at 70 ° C 25. Remove the elution buffer from the membrane into fresh tubes Add 12. 5 11g carrier tRNA to each RNA sample and extract once wtth an equal volume of phenol.chloroform. 26. Add 2 . 5 vol. of ethanol. Collect the RNA precipttate and wash 1t as in steps 1-3. 27 . Punfy full-length RNA on a denaturing polyacrylamide gel (see Volume I, Chapter 1, Protocol 8) .

Cleavage of modified RNA 28. Dtssolve each of the modified RNAs from steps 27 in 20111 of 1 M aniline 1n 0 . 3 M sodium acetate, pH 3 .8 . Incubate for 20 min at 60 °C in the dark. Similarly treat samples of the control RNAs not used to form polyadenylation complexes (step 13). 29. Add 1.4 ml of butan-1-ol. 30. Vortex and recover the RNA precipitate by centrifugation in a microcentnfuge for 1 5 min 31 . Redissolve the RNA in 150111 of 1% SDS and repeat steps 29 and 30. 32. Wash the RNA pellets by adding 1 ml of 70% ethanol and centnfugmg in a microcentnfuge for 5 min. 33. Dissolve the RNA in 5 IJI of urea loading buffer 34. Determine the amount of radioactivity recovered by Cerenkov counting . 35. Dilute aliquots of each sample (containing equal amounts of radtoacttvtty) wtth urea loading buffer, heat for 90 sec at 90 °C and load on a 12% denaturing polyacrylamide gel (sequencing gel)'. 36. Run the gel until the bromophenol blue marker IS 1 5 em from the bottom and detect the RNA by autoradiography.

Contmued 18

Elmar Wahle and Walter Keller

Protocol 5 . Continued • Store opened bottles of DEPC 1n a refrigerator for no more than one week . Store hydraz1ne 1n a closed container with some desiccating material 1n a fume hood at room temperature. c Redistill the aniline under nitrogen and store 1t in aliquots at - 20 ° C in the dark. Be careful: aniline is toxic and combustible. d Deionize the formam1de with a mixed-bed ion-exchange resin and store it 1n the dark at -20 °C. • T he extent to wh1ch prote1ns contained 1n the complex are transferred to the DEAE membrane together w1th the RNA IS unknown. Any protein transferred to the membrane and eluted in steps 21-24 w1ll be removed by the phenol:chlorolorm extraction in step 25. ' If the sample in urea loading buffer is heated lor too long, urea may precipitate Red1ssolve it by adding a small amount of water 0

A few experimental points should be noted with respect to the modificationinterference procedure (Protocol 5): (a) The procedure does not tolerate much non-specific degradation of RNA since this will generate bands in the sequence ladder that do not stem from a niline cleavage of modified nucleotides. Therefore, exercise care to avoid RNase contamination (see Volume I, Chapter I, Section 1.1). The shorter a substrate RNA is the easier it is to use in this protocol since short molecules are obviously less susceptible to non-specific degradation than long molecules. RNase inhibitor from placenta may also be used (see Protocol 6, footnote b). (b) The RNA substrate used for modification-interference can also be 5' endlabelled with [-y-32p] ATP and polynucleotide kinase instead of with RNA ligase. However, it has been reported that 5' -labelled RNA does not give clean sequencing ladders with pyrimidine modifications (15). (c) If, after analysis, the extent of modification is found to be too high or too low, vary the incubation times for the modification reactions (see Protocol 5, steps 5 and 9). (d) In Protocol 5, the sequence ladders w which the RNA from the specific polyadenylation complex is compared are generated from substrate RNA that has never been incubated with nuclear extract. These control ladders may also be generated from RNA isolated from non-specific complexes. For unknown reasons, cleavage at G residues is more efficient with RNA that has been incubated with extract and eluted from the non-denaturing gel. The best control is thus to use both substrate RNA without incubation and RNA from non-specific complexes. Figure 3 shows the results of a modification-interference experiment that was carried out with purified CPSF rather than nuclear extract. The nucleotide sequence of the substrate RNA can be read in the outer lanes (uncomplexed 19

1: 3' end-processing of mRNA A+G C+U ,-----, ,-----.,

-

--•-

----..

A-

A-&__

c

-- c

s:-·= ~

-- u

-

a:~ •G •G

-

A -A-A-G

l!l

--

C•

--

u

-

i 2 34 Figure 3. Modification-Interference analysis of the btnding of CPSF to an AAUAAA-conta1ning RNA. A ' precleaved' RNA (65 ntl. containing the upstream polyadenylation s1gnal of the adenovirus L3 s1te and endtng close to the natural polyadenylation site, was modif1ed at either pyrimidine or purine res1dues (see Protoco/5). Complexes were formed between the modified RNA and CPSF purified from calf thymus (see Section 3 .2.3 ; ref. 23). Complexed and free RNA were further analysed as described in Protocol 5. Lanes 1 and 4 contain uncomplexed RNA, lanes 2 and 3 contain RNA from the complex. All SIX bands corresponding to the AAUAAA sequence are strongly decreased in the RNA purified from the complex, indicating that the modification of any of these nucleotides interferes with CPSF binding. In contrast, cleavage of the three nucleotides Immediately following the AAUAAA s1gnal is enhanced tn the complexed RNA. Reproduced from ref. 9.

RNA). The inner lanes contain RNA that was isolated from the complex and show that each of the bands corresponding to the AAUAAA sequences is strongly decreased, indicating that the modification of any of these six nucleotides inhibits the binding of CPSF. No other band is decreased and thus no other nucleotide is critical for binding. 20

Elmar Wahle and Walter Keller In a variation of ProtocolS, the modification-interference method can also be used to identify the nucleotides involved in the cleavage and polyadenylation reactions that follow complex formation. To do this, rather than isolating RNA from polyadenylation complexes separated in native gels, carry out the chosen processing reaction (Section 2.5) with the chemically modified RNA and then separate the cleavage products or polyadenylated RNA from unreacted substrate RNA using denaturing gel electrophoresis (15). Treat these RNAs according to Protocol 5 to analyse which nucleotides are essential for processing. For obvious reasons, polyadenylated products and 5 ' -cleavage products can only be analysed with 5' end-labelled RNA which may not always be a good substrate (see above).

2.5 Assay of cleavage and polyadenylation in nuclear extracts 2.5.1 An overview Endonucleolytic cleavage of the substrate RNA in vivo and in vitro is very rapidly followed by poly(A) addition. Thus, the cleaved intermediate is not normally present in detectable quantities. However, there are two major reasons why it is important to find conditions whereby cleavage can be separated from polyadenylation in vitro. First, if one wants to study each of the two reactions, in terms of their RNA sequence and protein factor requirements or their biochemical mechanism, one has to have separate assays for each of them. Second , there is no mechanistic dependence of polyadenylation on the cleavage event and no strict requirement for a particular distance between the AAUAAA sequence and the 3' -end that is to be polyadenylated. Thus, polyadenylation can occur independently of cleavage by simple AMP addition to the 3 ' -end of an RNA, even if this RNA is a perfect substrate for cleavage. Consequently, the fact that an RNA has been polyadenylated does not imply that cleavage has occurred. Therefore, one cannot use polyadenylation as a coupled assay for cleavage; a direct assay is necessary. Nevertheless, it is sometimes advantageous to study coupled cl1~avage and polyadenylation in vitro. This is described in the following section. Modifications to this basic method that allow the separate assay of cleavage and of polyadenylation as well as of non-specific polyadenylation are described in Sections 2.5.3, 2.5.4, and 2.5.5, respectively. 2.5.2 Coupled cleavage and polyadenylation Protocol6 describes how cleavage and polyadenylation are assayed in a nuclear extract. Alternatively, purified or partially purified processing factors can be used in place of the nuclear extract. ln these cases, include commercial creatine kinase at 10 11g/ ml in the reaction. This may be necessary to maintain the ATP concentration since the endogenous enzyme of the extract may be removed during the course of protein purification, while ATPases are likely to contaminate partially purified factors. Also include Nonidet P-40 (0.01 07o) 21

1: 3' end-processing of mRNA

and RNase-free BSA (0.2-0.5 mg/ ml) to stabilize purified factors which are used at low protein concentrations. Protocol 6 . Coupled cleavage and polyadenylation in nuclear extract

Equipment and reagents • Hela cell nuclear extract. radiolabelled substrate RNA (1 00-1000 c.p.m./ fmol) and reagents for formation of polyadenylation complexes as m Protocol 2 • Proteinase K ( 10 mg/ml in water) • 2 x proteinase K digestion buffer (0.2 M Tris- HCI, pH 7.5, 25 mM EDTA, 0.3 M NaCI, 2o/o SDS)

• Phenol:chloroform (1:1) and reagents for ethanol precipitation of RNA (see Protocol 5) • Denaturing polyacrylamide/urea gel, formamide loading buffer, and TBE buffer lsee Volume I, Chapter 1, Protocol B). plus appropriate electrophoresis equipment • RNase inhibitor from placenta (Pharmacia 'RNAguard')

Method 1. Using 10-60 fmol of substrate RNA, set up a 25 1-11 processing reaction as in Protocol 3, steps 1-48 • Include 4-5 units of RNase inhibitor to minimize RNA degradation (optional)b_ 2. Incubate the mixture for at least 1 h at 30 °C to allow cleavage and polyadenylation to occur.

3. Terminate the reaction by the addition of 100 1-11 of 2 x proteinase K digestion buffer, 21-11 of 10 mg/ml proteinase K, 21-11 of 2. 5 mg /ml tRNA and 71 1-11 of water. Incubate for 30 min at 30 °C. 4. Extract the mixture once with an equal volume of phenol:chloroformc.d. 5. Precipitate the RNA by the addition of 500 1-11 of ethanol. Collect the precipitate by centrifugation, wash it with 70% ethanol, and dry it (see Protocol 5. steps 1-3). During these steps, monitor the recovery of RNA with a hand-held Geiger counter. 6. Dissolve the RNA in 31JI of formamide loading buffer, boil it, and chill it on ice. Load the sample on a denaturing polyacrylamide gel". 7. Run and autoradiograph the gel to detect the RNA (see Protocol 3 ). • If the nuclear extract is replaced by purified factors, or if smaller volumes of extract are used, the incubation volume should be made up to one-half of the total reaction volume with buffer D (see Protocol 1 ). If either the nuclear extract or the purified factor samples contain higher concentrations of KCI, buffer D lack1ng KCI can be used to make up the volume. The final KCI concentration in the processing reaction should not be much higher than 50 mM. Note that the amount of Mg2 ' Introduced into the reaction with nuclear extract or protein fractions can vary (see Section 2.3) so some adjustment may be necessary. b Ribonuclease inhibitor from placenta inactivates RNases by forming a tight complex with them. This activity depends on the presence of DTT. Make sure that the DTT is not too old and has been stored under appropriate conditions (see Prorocol 1 ). If the inhibitor is inactivated, it will release any bound RNases which may then degrade the substrate RNA .

Continued

22

EJrnar Wahle and Walter Keller

Protocol 6. Continued A large white mterphase after phenol:chloroform extractton is due to PVA If it is too large to permtt efftctent separation of the phases, mcrease the volumes of the aqueous phase and of the phenol:chloroform mixture and increase the centrifugation time. • When the protem content of the reaction mtxture is low, the extraction can be omitted and the RNA can be prec1p1tated directly with ethanol. This has to be tested under the particular conditions used. In this case it is important that not more than 2.5 vol. of ethanol are used for precipitation of the RNA. Otherwise, PVA will also precipitate. • Choose the acrylamide co:mtent of the gel and the runntng time accordmg to the stze of the precursor RNA and the expected products (see Figure 4 for an example). c

2 .5.3 Cleavage of RNA substrates in vitro Two different modifications of the coupled reaction (see Protoco/6) are widely used to block polyadenylation and thus allow the cleavage reaction itself to be studied. One method is to omit Mg2 + (except the Mgh introduced with the nuclear extract) and to include l mM EDTA in the reaction. This inhibits poly(A) polymerase but permits cleavage. Not all precursor RNAs are processed well under these conditions. EDTA inhibits nucleases in the extract so that the downstream cleavage product may also be detected. The other method is to replace ATP with one of two different classes of analogues. Chain-terminating analogues lack a 3 1 -hydroxyl group. Thus, only a single nucleotide can be incorporated at the poly(A) site so that the cleaved RNA can be detected. ATP analogues with a non-hydrolysable a-~ bond [e.g. (a-~ methylene)A TP] cannot be used as substrates for polyadenylation so that the 5' -cleavage product is not extended at all. ATP analogues are easier to use than EDTA. The· preferred compound is 3 1 -deoxy-ATP (cordycepin triphosphate). Thus, to examine the 3 1 -cleavage of RNA, replace ATP in Protocol 6, step I, with 0.5 mM 3 ' -dATP. However, note that the nuclear extract may still contain ATP, especially if it has not been dialysed. This endogenous ATP competes with 3 '-dATP so that some elongation may still be observed. The downstream product of RNA cleavage is rapidly degraded and cannot be detected. The cleavage reaction must be incubated at 30 oc; it does not work at 37 °C. 2 .5.4 Polyadenylation of RNA substrates in vitro Polyadenylation can be measured independently of cleavage simply through the use of a 'precleaved' substrate RNA that ends at, or close to, the natural poly(A) site, downstream of the AAUAAA sequence. For 'precleaved' RNA, there is no strict requirement either for the distance between AAUAAA and the 3' -end or for the sequence at the end itself. Use any convenient restriction site in the recombinant plasmid close to the AAUAAA sequence to prepare the template for run-off transcription (see Volume 1, 23

1: 3' end-processing

of mRNA

Chapter I, Section 2.1 ). The natural distance between AAUAAA and the poly(A) site is 15-30 nt. The method for assaying polyadenylation in the absence of cleavage follows Pro10col 6 except that a 'precleaved' substrate RNA is used. ATP is included as the substrate for poly(A) polymerase. Polyadenylation occurs at least as efficiently at 37 oc as at 30 oc. An incubation time of 30 min is usually sufficient. The specific polyadenylation reaction is critically dependent on Mg2 - ions. Lower concentrations of Mg2 • (and also of KCl) may stimulate polyadenylation but, at least with purified factors, they also reduce the specificity (i.e. AAUAAA-dependence) of the reaction. The use of a substrate RNA with a point mutation in AAUAAA is an imponant control. Dialysed and non-dialysed extracts differ in their Mg2 - concentration (see Section 2.3). In any case, the Mg2 + content of the extract is unlikely to be known accurately. A titration of Mg2+ with a wild-type and a mutant substrate may thus be useful. 2.5.5 Non-specific polyadenylation As discussed in Section 2.5.4, the dependence of polyadenylation on the AAUAAA sequence in the substrate RNA is influenced by the reaction conditions, especially by the presence of Mg2+. If Mg2+ is substituted by Mn2 +, the specificity is lost and poly(A) polymerase can polyadenylate any RNA, independent of the AAUAAA sequence and of CPSF (Section 2.2). To carry out this non-specific reaction, replace MgC1 2 in Protocol 6, step I, with an amount of MnC12 equimolar to the amount of ATP used (0.8. mM). As a substrate, use an RNA with a point mutation in the AAUAAA sequence or any other RNA that does not contain the polyadenylation signal. The non-specific polyadenylation reaction is useful for checking the quality of RNA 3 '-ends (see Section 2.4.4) and as an assay for poly(A) polymerase. Protocol 8 (Section 3.2.4) provides a modification of the non-specific polyadenylation reaction that is panicularly useful for the latter purpose. 2.5.6 Interpretation of the data Figure 4 shows typical data from an experiment in which the polyadenylation reaction (Figure 4A) and the cleavage reaction (Figure 48) were studied separate!}

as described in Sections 2.5.3 and 2.5.4. Since the poly(A) tails of mRNAs are not of uniform size, but show a s1ze distribution around a mean of approximately 200 nt, poly(A)- RNA forms a smear in the gel (Figure 4A), migrating more slowly than the precursor RNA. The appearance of the polyadenylated product in a coupled cleavage/polyadenylation assay (Figure 1B) is the same as in a polyadenylation assay with a precleaved substrate RNA (Figure 4A). However, as pointed out in Section 2.5.1, the poly{A)- RNA generated in a coupled assay may contain a mixture of molecules that have been correctly cleaved and then polyadenylated and others that have received 24

Elmar Wahle and Walter Keller

A

8 309

poly(A)l

242_ 238 21720L 190180 160141-

pre

122-

1 2

110-

M

2

Figure 4 . Cleavage and polyadenylation assays in HeLa cell nuclear extract. (A) A polyadenylation assay was carried out 1n nuclear extract with the precleaved RNA described in Figure 3. Lane 1, precleaved RNA (pre) was incubated without nuclear extract. Lane 2, RNA after Incubation with extract. Products, Poly(A). were analysed on a 12% denaturing gel. No size markers were included in this gel fBI A cleavage assay was carried out in HeLa cell nuclear extract in the presence of ddA TP with the L3 RNA described in Figure 1 Lane 1, incubation without extract. Lane 2, incubation w1th extract RNA was analysed on a 6% denaturing gel. 'Pre' indicates the RNA substrate. The arrowhead Indicates the upstream cleavage product. Lane M contains DNA size markers With the sizes (nt) 1nd1cated on the left

poly(A) at their original 3 '-end without having been cleaved. Note that the latter reaction is also dependent on the AAUAAA sequence in the substrate RNA. It is thus different from the non-specific polyadenylation reaction described in Sections 2.5.5 and 3.2.4. The cleavage reaction shown in Figure 4B was performed with dideoxy-ATP (ddATP) as the chain terminating analogue. (Note, however, that 3 '-dATP is more effictent than ddATP.) Since the cleavage product migrates faster than the substrate among all the fragments generated by non-specific nuclease action, the specificity of the cleavage reaction has to be verified not just by the size of the product but also by its dependence on the correct processing signal sequences. As a control for the cleavage reaction, use a precursor RNA with a point mutation in the AAUAAA sequence. 25

1: 3' end-processing

of mRNA

3 . Polyadenylation and 3'-cleavage factors 3.1 Overview of processing factors The reactions of 3 ' -cleavage and polyadenylation are carried out by at least five, possibly more, factors (see Section I ). Of the two or more cleavage factors, only one, a heterOLrimer called CStF or CF1, has been purified to homogeneity (16, 17), and cDNAs encoding one of its subunits have been cloned (18). The cleavage factors can be assayed only by reconstitution of the entire cleavage reaction (see Section 2.5.3) since no partial reactions have been identified that are catalysed by these components. Poly(A) polymerase has been purified to homogeneity (19, 20) and cDNAs encoding the enzyme have been cloned (21, 22). Poly(A) polymerase is most easily assayed by the non-specific polyadenylation reaction as described in Section 3.2.4. The specificity factor CPSF has been purified to homogeneity (23). It can be assayed by the specific polyadenylation assay when it is supplemented with purified poly(A) polymerase or by specific binding to AAUAAA-containing RNAs (Section 3.2.5). A poly(A) binding protein that causes poly(A) polymerase to elongate oligo(A) tails rapidly has also been purified to homogeneity (24; E. Wahle, unpublished data). This factor can be assayed by its activity in stimulating specific polyadenylation or by its specific poly(A) binding activity. For details, consult ref. 24. A detailed description of protein purification procedures is beyond the scope of this article. However, Section 3.2 gives some general advice and describes methods for separating poly(A) polymerase from CPSF and for assaying these two factors during purification .

3.2 Purification of processing factors 3 .2.1 General considerations Before a protein purification is undertaken, some consideration should be given to the choice of starting material. The almost exclusive use of HeL. cell nuclear extract for RNA processing experiments does not reflect a particula1 wealth of processing factors in HeLa cells, but merely the fact that their Jo,, level of nucleases permits the processing reactions to be readily observed. For purification of processing factors, HeLa cells have some disadvantages, ont of which is cost. If a protein has to be purified 10 000-fold with a yield of 10117o 5 g of extract protein have to be processed to yield 50 J.Jg of homogeneou material. This corresponds to the nuclear extract from several hundred grams of HeLa cells. Much more than this is consumed during the development O• the purification procedure. These quantities may go beyond the fmancta capacities of many laboratories. Also, HeLa cell nuclear extract does not appeal to be particularly rich in 3 ' -processing factors. Both poly(A) polymerase and CPSF are as much as ten-fold more abundant in calf thymus extract than in nuclear extract from the same amount of HeLa cells. Thus, whereas I kg of 26

Elmar Wahle and \\'alter Keller calf thymus yield~ approximately 0.5 mg of poly(A) polymerase (19, 20), onl} 50 11g would be expected from I kg of HeLa cells. Even this is an optimistic estimate since the small quantities present in HeLa cells would make the purification more difficult. If, despite these potential problems, HeLa cells are chosen as the source for the purification of protein factors, one should remember that the standard nuclear extract preparation (see Protocol I) has not been optimized for the factor of interest. Significant quantities of this particular factor may have leaked from the nuclei into the C)'10plasm during cell lysis or the salt concentration may not be optimal for its extraction from the nuclei.

3.2.2 Separation of poly(A) polymerase and CPSF from a HeLa cell nuclear extract Protocol 7 describes the separation of poly(A) polymerase from CPSF by DEAE

column chromatography of HeLa cell nuclear e:xtract. Poly(A) polymerase is found in the flow-through along with at least one cleavage factor, whereas CPSF binds to and is eluted from the column along with at least one other cleavage factor. Assays for these factors are given in Sections 3.2.3 and 3.2.4. Obviously, the same fractions may also be assayed for cleavage activity (see Section 2.5.3).

Protocol 7 . Fractionation of Hela cell nuclear extract by DEAE chromatography Equipment and reagents • Hela cell nuclear extract (see Protocol 1)" • Chromatography column• fraction collector • Penstaltic pump lopt1onaW, and conductivity meter • OEAE chromatography matr1x (OEAESepharose (Pharmacial or equivalent)

• 1.0 M OTT (see Protocol 1) • Starting buffer (50 mM Tns-HCI. pH 7 .9. 75 mM KCI, 0.5 mM EDTA, 0.5 mM OTT". 1O'lb glycerol) e start1ng buffer without KCI • Elution buffer (as start1ng buffer. but conta1n1rog 0.5 M KCI)

Method 1. Make a slurry of DEAE-Sepharose in starting buffer and pour the column. Equilibrate the column in starting buffer until the conductivity and pH of the washings are identical to those of the starting buffere. 2 . Set up the column in a coldroom and carry OLit the rest of the procedure as close to 0 °C as possible. 3. Dilute the Hela cell nuclear extract with ice-c.old starting buffer without KCI until its conducttvity equals that of the s1art1ng buffer' 4 . Pump the diluted nuclear extract onto the column at about 1 column vol./h. Load approximately 10 mg of protein.ml colurnn vol.

Continued 27

1: 3' end-processing of mRNA

Protocol 7 . Continued 5. Collect the flow-through, excluding as much as poss1ble of the buffer pre· cedmg the protein peak. A fraction collector is helpful but not necessary. 6. Wash the column with one volume of starting buffer and combine the wash with the flow-through. Precipitate the proteins with ammonium sulphate (step 9). 7 . Elute the column with elution buffer. The beginning of the salt step can be identified by the mcrease in conductivity or in protein concentration. D1rect activity assays may not be possible at this stepll. 8 . Dialyse the eluate against starting buffer. 9. To the combmed flow-through and washings add solid ammonium sulphate to 80% saturation (0.5 16 g of ammonium sulphate per ml). Stir the mixture on ice until the salt has dissolved and then for at least one additional hour.

10. Centrifuge at 18 000 9max for 30 min. 11. Dissolve the precipitate in the smallest volume of starting buffer possible and dialyse against starting buffer. 12. After dialysis, check the conductivity to make sure that most of the ammonium sulphate has been removed. 13. Assay the two fractions (flow-through and eluate) for poly (A) polymerase and for CPSF (see Sections 3. 2.4 and 3.2.51 • Not dialysed (see Protocol 1, step 11 ). Dialysed extracts are less active for cleavage factors • Choose a column large enough to accommodate the protein 1n the nuclear extract (see step 41. The length should be approximately 5 times the diameter • A fraction collector with a UV monitor is convenient but can be misleading. Optical density at 280 nm may indicate the presence of protem or nucleic acids, or light scattering by particulate material. A d1rect protein assay 125) is more reliable. d Add DTI just before use from the 1.0 M stock solution • The equilibration IS faster if the DEAE slurry is f~rst titrated to the des~red pH before 1t is poured into the column. (Do not use a magnetic stirrer to Stlf the slurry as this may destroy the matrix. I ' D1alys1s of the nuclear extract instead of dilution is also possible (but see footnote a) In this case, ammonium sulphate precipitation is not necessary if only the peak flow· through fractions are collected without d1lution by the washmgs. ' With a now-through UV monitor, collect1on of the enure peak in a smgle fraction is poss1ble. If the peak has to be identified by protem assays or conductivity measurements, collect fractions of approximately one quarter of the column volume.

The behaviour of any of the processing factors during chromatograph' depends on a number of variables, and so the separation may not be complete If this is the case, try varying the salt concentration of the starting buffer o reducing the amount of protein loaded on the column. If the DEAE column is intended for a simple separation of poly(A) polymerase and CPSF, a step elution as described in Protocol 7 (step 7) is adequate. I DEAE chromatography is used as the first step in a more complete purification of any processing factor, the column should be eluted with a gradient (e.g. 28

Elmar \Vahle and \\'alter KeJler

10 column vol. from starting buffer to elution buffer). Under these conditions, CPSF and a cleavage factor elute in overlapping peaks around 0.15 M KCI.

3.2.3 Purification of processing factors from calf thymus Although a high nuclease content generally does not permit the observation of RNA processing reactions in tissue extract, polyadenylation is an exception. This is due to the fact that the poly(A) tails are quite resistant to degradation. If the precursor RNA is shorter than the poly(A) tails (i.e. shorter than 200nt), any polyadenylated product, however badly degraded it may be, will stiLl migrate more slowly than rhe precursor RNA in a gel and will thus be distinguishable from the degraded material. As long as it retains a single labelled nucleotide, it wiU be detectable by autoradiography. Very little RNA remains after incubation in the extract, but, in the course of the purification of processing factors, the situation improves with every column. Since cleavage assays are much more sensitive to RNases, however, they cannot be carried out with tissue extracts. Protocol 7 can be easily adapted as the first step in the purification of poly(A) polymerase and CPSF from calf thymus (23). ln this case, a total extract prepared in a Waring Blendor (20) is used as starting material rather than nuclear extract. The main practical problem is the presence of large amounts of particulate material in the calf thymus extract. This material cannot be removed by centrifugation. The most convenient and generaUy applicable way of removing it is to appl)' the turbid extract directly to the DEAE column. In this case, a short, rather wide column packed with a material with good flow properties [e.g. DEAE-Sepharose Fastflow (Pharmacia)] is important. When the column becomes clogged, stir up the top layer and continue loadmg the sample. This procedure may have to be repeated several times during loading. The column will not clog during washing and elution. A major portion of the particulate material passes rapidly through the column. Apparently through a gel filtration effect, it stans to appear in the flow-through after only one-half of a column volume of buffer has passed through, '"hereas poly(A) polymerase activity and total protein increase significantly only after almost one column volume. Thus, poly(A) polymerase is partially separated from the particulate material. The enzyme can be precipitated by ammonium sulphate (at 5007o saturation). After dialysis against phosphate buffer and dilution, theremaining particles aggregate and can then be removed by centrifugation before the enzyme is applied to a Biorex column (Bio-Rad). Six additional columns are needed after the initial DEAE column to bring the protein to homogeneity. For details of the purification procedure, consult ref. 20. Note that the enzyme as described initially (19, 20) had suffered partial proteolysis (21, 22). Undegraded enzyme can be obtained by essentially the same procedure, except that the protease mhibitors PMSF (0.5 mM), leupeptin (0.4 ~g, ml), and pepstatin (0.7 ~g/rnl) should be included throughout all purification steps (E. Wahle, unpublished data). CPSF bound to the DEAE column and eluted with a salt gradient will be Largely, although not entirely, free of particulate material. It~ purifi,:ation 29

1: 3' end-processing

of mRNA

to near homogeneity requires five additional steps (23). A third factor, poly(A) binding protein II, can be isolated from the DEAE-bound calf thymus fractions by three additional steps as described in ref. 24. The separated processing factors are assayed as described in the following sections.

3.2.4 A convenient assay for poly(A) polymerase The assay described tn Protocol 8 is useful for monitonng the punfication of poly(A) polymerase because it is very fast and quantitative. In a non-specific, Mn 2 - -dependent reaction, the enzyme adds radiolabelled AMP residues from [ a:--32P] A TP to an unlabelled poly(A) primer. The radiolabelled reaction product is separated from unincorporated A TP by TCA precipitation and filtration. As an alternative to TCA precipitation, the incorporation of AMP can be quantified by adsorption to DEAE paper as described in ref. 20. Th1s is necessary if short ohgonucle01ides are used as primers. The non-specifk extension of poly(A) has the additional advantage that this polynucleotide, which is both the primer and the product of the reaction, is insensitive to many nucleases. Thus, this assay can be used in extracts of almost any tissue. Commercial preparations of homoribopolymers can have substantial proportions of their 3 1 -ends blocked by phosphate groups. These polymers are inactive as primers for poly(A) polymerase. Unless these phosphates are 2 1 -3 cyclic phosphates from alkaline hydrolysis of the poly(A), they can be remove-. "' ith calf intestinal alkaline phosphatase. This will activate the polynucleotid.. as a primer (20). However, it is easier and cheaper to try a different batch o • the poly(A) primer. Note that the activity of the enzyme with a given bate! of primer depends not only on the extent to which the 3 ' -ends are blocked but also on the length of the primer, since, at a given concentration of poly(A), the length determines the concentraction of 3 1 -ends. Results obtained with different batches of poly(A) are, therefore, not necessarily comparable. Non-specific polyadenylation can also occur in the presence of Mg2 + ions. However, the reaction is much less efficient due to a lo" affinity of the polymerase for the 3' -end of the RNA primer (20). Protocol 8 . Measurement of poly(Al polymerase activity by a TeA-precipitation assay Equipment and reagents • Vacuum filtration dev1ce su1table for 24 mm diameter filters' (e.g Schleicher and Schuell, SartoriUS, Mlllipore)

• 25 mM DTI td1luted from a 1 M stock solution; see Protocol 11

• 0.1 M ATP (see Protocol 2)

• 2 x polyadenylatlon buffer (20% glycerol 50 mM Tns HCI, pH 8.8°, 0.1 mM EDTA , 0.02% Nonidet P-40, 0 .4 mg/ml BSAl

e 1 M KCI

• la-32P]ATP

e 25 mM

MnCI2

(Amersham: 400CIImmo1J·

Contmued 30

EJmar \\'ahle and Walter Keller Protocol 8 . Continued e Poly(A) (Boehnnger Mannhe1m, Pharmacia , or Sigma)d

e Wash solution (0.5% TCA, sodium pyrophosphate)

• Phenol:chloroform I 1 1 J

• Glass f1bre filters !Whatman GFJC, 24 mm d1ameterl

• TCA solution (1 0% (wlv ) tnchloroacet1c ac1d. 0 1 M sod1um pyrophosphate )

10mM

Method 1 . M1x (et-32P] ATP, 0.1 M ATP. and water to prov1de a 12.5 mM 3 2P-Iabelled ATP working solution at 20-100 c .p.m./pmoL Spot 1 Ill onto a GF/C filter and count it in a scintillation counter. Calculate the specific radioactivity, ignoring the contribution of the 3 2P-Iabelled ATP to the total A TP concentration. 2. Make a stock solution of poly(AJ; d1ssolve commercial poly(A) in water at approximately 10 mg.ml, extract this once w1th phenol:chloroform and dialyse the solution exhaustively against water. Determine the exact concentration from the A 258 le258 = 9800; the extinction coefficient refers to the concentration of the AMP monomers).

3. In a microcentrifuge tube in an ice bath, set up a reaction mixture containing the following (sufficient for 10 assays): • 2 x polyadenylation buffer

125 (.II

e 1 M KCI

10(..11

e 25mM MnCI 2

5(.11

e 12.5mM (et- 32P]ATP (step 1)

10 (.II

• 5 mg/ml poly(A)e

15 (J(

e 25mM OTT

51)1

93. 5(.11 • H2 0 4 . Dispense 25 (.II aliquots of this mixture into 1. 5 ml microcentrifuge tubes on ice. To each tube, add an aliquot (0. 5-31)1) of the column fraction or enzyme preparation to be assayed' Include a tube with column buffer replacmg the enzyme to determine the background radioactivity and a sample of known poly(A) polymerase activity as a positive control, if available. 5 . Incubate the tubes at 37 °C for 10 min or longer. depending on the activity of the fract1ons . 6 . Stop the reaction by adding 100 (.II of TCA solution Vortex and place on ice. 7 · Soak the glass fibre filters m wash solut1on. place one on a vacuum filtration unit and apply the vacuum 8 . Fill the f~rst assay tube w1th wash solut1on from a squeeze bottle. Do this gently. otherw1se you may splash the contents of the tube. Pour the contents of the tube on the filter. 9 . Wash the tube out tw1ce with wash solution, allowing the washmgs to pass through the filter

Continued 31

1: 3' end-processing

of mRNA

Protocol 8 . Continued 10. Finally, wash the filter and the filtration device w1th a few mlllihtres of wash solut1on followed by ethanol.

11. Number the filter with a soft penc1l and place 1t under a heat lamp. 12. Filter all other samples in the same manner and count the radioactivity retamed on each filter m a scintillation counter.

13. Calculate the amount of poly(AI polymerase act1vity. One unit of enzyme actiVity mcorporates 1 pmol AMP per min. • A unit holding a single filter is much eas1er to use than those holdmg many filters. Also, since filtration through glass fibre filters IS very rapid , one does not gain time by performing several filtrations at once ' Make up a 1.0 M stock solution of Tns buffer and adjust the pH at room temperature. Use this stock to make up the polyadenylauon buHer without readJUSting the pH. Th1s will result m a pH of 8.3 under reacuon conditions. the opt1mum for poly(AI polymerase in th1s assay (20). Since the 32P-Iabelled ATP is moxed with unlabelled ATP, a high specific activ1ty is not necessary. Also, the labelled A TP does not have to be fresh - The average size of the poly(A) varies from batch to batch, from less than 100 and up to several hundred nucleotides. The only commerical product of defined size that the authors are aware of is A ,, 2 _, 11 (Pharmacia). • Change the volume of this reagent according to the concentration of your particular stock solution. Also, the amount given is only a suggestion. The concentration of primer required to saturate the enzyme depends on the length of the polynucleotide. the K,. of polyiA) polymerase for the 3'-end of the primer 1s approximately 41JM under these conditions (201 1 Dialys1s of enzyme fractions pnor to the assay os normally not necessary s1nce the volume added IS small and the react1on is not very sens1t1ve to the salt concentration. If desired, the salt concentration 1n all assays can be made odent1cal by vary1ng the amount of KCI added in step 3 to take into account the KCI concentration in the sample to be assayed.

Under conditions of primer saturation, HeLa cell nuclear extract should ba' e 500-1000 units of poly(A) polymerase/ mg protein by this assay. Calf thymus extract should have 1500-2000 units/ mg. Poly(A) polymerase purified to homogeneity has in excess of 107 units/ mg (20). Poly(A) polymerase can also be assayed by the specific polyadenylation reaction utilizing RNA substrates containing the AAUAAA polyadenylation signal (see Protoco/6). However, this Mgl+ -dependent reaction also requires the addition of CPSF from the DEAE-bound fractions. The assay is not very convenient for monitoring the purification of poly(A) polymerase.

3.2.5 Assay of CPSF Pure or partially purified CPSF can be assayed by the specific polyadenylauon reaction (see Protocol 6) when it is supplemented with poly(A) polymerase. Alternatively , complex formation with a radiolabelled RNA containing an AAUAAA sequence may be assayed, essentially as descnbed in Protocol 2. In each case, the nuclear extract is replaced by the CPSF fraction and the 32

Elmar Wahle and Walter Keller

reaction volume is made up as necessary with buffer D (see Protocol I). The complex formation assay {see Protocol 2) is modified as follows: • ATP and creatine phosphate may be omitted since binding of purified CPSF to the AAUAAA sequence is A TP-independent. • Samples are incubated for IOmin at 37 oc. • The CPSF -AAUAAA complex is sensitive to heparin. Thus, neither heparin nor tracking dyes are added. Tracking dyes are run in an empty lane. • The samples are loaded directly onto the native gel while it is running. (Be careful: do not electrocute yourself.)

4. Analysis of polyadenylation in vivo In vivo analyses are frequently used to determine sequence requirements for polyadenylation. Whereas the requirement for the AAUAAA sequence and for the downstream elements has been seen both in vivo and in vitro, upstream sequences necessary for 3' end-processing have so far been demonstrated only m vivo (re' iewed in ref. 26). Transient expression of transfected plasmid DNA is generally used as an assay. RNA is isolated from the cells {see Volume I, Chapter I, Section 3) and analysed by Northern blotting, nuclease Sl analysis, or RNase protection {see Volume I, Chapter 2, Section 3). Polyadenylation is an essential step in mRNA synthesis. The result of a mutational inactivation of the polyadenylation site is not generally the accumulation of the precursor RNA but a strong reduction in the concentration of the mature RNA. Unprocessed precusor appears to be degraded. Since the result of such a mutation is the absence of the RNA of interest, it is essential that the experiment contains the proper controls to ensure that this absence is not the result of a non-specific effect. The transfection efficiency, RNA extraction, blotting, etc. should all be controlled for by the inclusion of an unrelated standard plasmid. However, the most useful control is to include a known strong polyadenylation site {e.g. from SV40 or adenovtrus) in all the experimental plasmid constructions downstream of the site that is being tested by mutation. An} RNA that fails to be processed at the upstream site under tnvestigation will be 'rescued' by the downstream site. The efficienq of the site being analysed can thus be directly compared to the internal standard site. Examples of this strateg} and experimental procedures can be found in refs 27 and 28.

References I. Wahle, E. and Keller, W. (1992). Annu. Re1•. Biochem.• 61, 419. 2. Moore, C. L. and Sharp. P. A. (1985). Cell. 41, 845. 3. Wahle, E. (1992). Bioessays, 14,113. 33

1: 3' end-processing of mRNA 4. Stauber, D., Soldati, D., Luscher, B., and Schi.lmperli, D. (1990). In Methods 111 enzymology. Vol. 181 (ed J. E. Dahlberg and J. N. Abelson), p. 74. Academic Press, San Diego. 5. Birnstiel. \1 L. and Schaufele, F. J. (1988) In Structure and function of major and mmor small nuclear ribonucleoprotein particles (ed. ~1. L. Btrnstiel). p. 155. Springer. Berlin. 6. Dtgnam, J. D., Lebovitz, R. ~1.. and Roeder, R. G. (1983). Nucle1c Acids Res. 11 , 1475 . 7. Lee, K. A. W. and Green, M. R. (1990). In Methods in enzymology, Vol. 181 (ed. J. E. Dahlberg and J. N. Abelson), p. 20. Academtc Press, San Diego. 8. Zerivitz, K and Akusjan i, G . (1989). Gene Anal Techn., 6, 101. 9. Keller, W. , Bienroth, S., Lang, K. M., and Christofori, G. (1991). £.\fBO J., 10, 4241. 10. Weiss, E. A., Gilmartin, G., and Nevins, J. R. (1991). EMBO J., 10, 215. I I. Moore, C. L., Skolnik-David, H., and Sharp, P. A. (1988). Mol. Cell. Bioi., 8, 226 12. Konarska, \1. M. (1989). In Methods in enz..vmofogy, Vol. 180 (ed J. E. Dahlber· and J. N . Abelson). p . 44-2. Academic Press. San Otego. 13. Humphrey, T., Christofori, G. , Lucijanic, V., and Keller,\\ . (1987). EMBO J 6, 4159. 14. Kramer, A. (1987). J. Mol. B1of., 196, 559. 15. Conway, L. and Wickem., M. (1987). EMBO J., 6, 4177. 16. Takagaki, Y., Manley, J. L., MacDonald, C. C . , Wilusz, J., and Shenk, T . (1990) Genes Dev., 4, 2112. 17 Gilmartin, G . and Nevin~>. J. R . (1991). '.fof Cell. Blot.• 11 , 2431. 18. Takagaki, Y, MacDona!. C C., Shenk, T .. and Manley. J . L. (1992). Proc. '\a1 Acad. Set. USA, 89, I.W3. 19. Tsiapalis, C. M., Dorson, J. W., and Bollum, F. J . (1975). J. Bioi. Chem., 250,4486 20. Wahle, E. (1991). J. Bioi. Chern., 266, 3131. 21. Raabe, T., Bollum. F. J ., and Manley, J. L. (1991). Nature, 353, 229. 22. Wahle, E., Martin, G., Schtlz, E., and Keller, W. (1991) EMBO J., 10, 425 1. 23. Bienroth, S., \\'ahl , E., Suter-Crazzolara, C., and Keller, \\ (1991). J Bioi. Chem.. 266, 19 768 24. Wahle, E. (1991). Cell, M, 759. 25. Bradford, M. M. (1976). Anal. Bwchem., 72, 248. 26. Proudfoot, N. (1991). Cell, 64, 671. 27 Iwasaki, K. and Temin, H. M. (1990). Genes Det:. , 4, 2299. 28. Russnak, R . and Ganem, D. (1990). Genes Dev. , 4 , 764.

34

Capping and methylation of mRNA YASUHIRO FURUICHl and AARON J. S H ATKIN

1. Introduction A 'cap' structure of the type m 7 GpppNmpNmp (Figure 1) is present at the 5 ' -terminus of almost all eukaryotic mRNAs (1, 2). Caps are formed on cellular mRNA precursors in the nucleus during the initial phases of transcription and before several other RNA processing events take place, including internal N6A methylation, 3 ' -poly(A) addition, and exon splicing. Cap structures with multiple methyl groups on the terminal guanosine residue (i.e. ml·2•7 GpppNm) are also present on the small nuclear RNA species (snRNAs) which apparently provide scaffolding for the various steps in mRNA splicing (3). With one possible excepuon (4), all capped cellular and viral mRNAs so far examined contain only a single methyl group on the terminal G residue, while the adjacent nucleotides are 2 1 -0-methylated to different extents, providing a 'basis for the following cap nomenclature: m 7GpppN {Cap 0), m 7GpppNm (Cap I) and m7GpppNmpNm (Cap 2). The methyl groups in Cap 0 and Cap I struc:tures are added in the nucleus while the additional 2 ' -0-methylation in Catp 2 is a cytoplasmic event, like the 2-amino methylations on snRNAs (5). In many lower eukaryotes, including yeast, mRNAs contain mainly Cap 0, whil.e higher organisms usually have more extensively methylated caps (1). It should also be noted that trypanosome mRNAs contain a trans-spliced, capped leader RNA with several modified residues as pan of the cap {6, 7), and in some mRNAs where base I is an adenine residue, ring methylation on the N6 position can also occur. Despite these vanations on the methylation theme, the important biological consequences of a cap structure appear to correlate only with the N-7 methyl group on the 5 -terminal G. For example, caps increase mRNA stability by protecting the mRNA against 5 1 -+ 3 1 exonucleolytic degradation (8). Unmethylated caps can also stabilize mRNA to a certain extent, but unless the 5 ' -termmal GpppN is converted lO m~GpppN, it is still capable of being hydro lysed to (p)pN, resulting in the subsequent destabilization of the decapped mRNA. Splicing accuracy and efficiency are both increased by the Presence of the 5' -terminal m1 GpppN (9, 10), possibly due to the involvement of nuclear cap-binding protein(s) ( II) analogous to the cytoplasmic cap-binding I

2: Copping and methylation of mRNA 0

CIIJ

NH~~•

I ~--)_),2 3 basel HAN N ,. 1's· ~ ~ ~ s~ -~CHz-0-P-0-P-0-P-0-HlC 0

-. 1

~

HIIO

I

I 0I 0I 0-

0

3' 0

I

0

2' 0-CIIJ

basc2

5'

=~-O-II2Cq 03'

l'

0

o--CHJ

I O=P- o-tR!\A I

chain)

0-

Figure 1. Cap structure of the type m7 GpppN"'pN~p (Cap 2) show1ng the m'-guanosir linked to the 5 '-end of the pnmary transcript via a 5 ' -5' phosphodiester bond The ribos.. moieties of the ftrst two nucleotides (w1th bases 1 and 2) of the primary transcnpt are 2 '-0· methylated. Both of these 2 '-0-methyl groups are absent in Cap 0 while in Cap 1 only th first nucleotide is 2'-0-methylated. Where the 5' nucleotide is an adenylate residue, the adenin (base 1 ) may be additionally methylated on the N8 position .

initiation factor e!F-4F that is required for eukaryotic protein synthesis (1 2) Interestingly, the donor of the methyl group(s) in the cap is an activated form of L-methionine, S-adenosylmethionine (AdoMet), and protein synthes s is also initiated with an activated L-methlonine intermediate, initiator methion) ,_ tRNA. For most cellular and \'iral mRNAs, capping and subsequent methylation occurs by the scheme illustrated in Figure 2. The 5 ' -terminal end of the nasce·1t RNA product of RNA polymerase II is fust modified by removal of the 5 phosphate by RNA triphosphatase to yield a diphosphorylated end (ppNpNpThls is then capped by GMP transferred from GTP by mRNA guanyl} !transferase (GpppNpNp-). Subsequent methylation then occurs in stages catalysed by RNA methyltransferases using AdoMet as the methyl donor to produce the 5' cap structure, m 7 GpppNmpNmp-. RNA capping does not occur on the products of RNA polymerase I and lii which synthesize rRNAs, and the tRNAs and 5S rRNAs, respectively. This indicates that the enzyme(s} involved in mRNA capping are associated with RNA polymerase II and/ or other component(s) of the transcription initiation complex (13). 36

Yasuhiro Furuichi and Aaron }. Shatkin

ppptrp~p --Nascent mRNA (primary transcr:i.pt) RNA triphosphatase

.:)C~) ~ G (5

Ado~trl \doHr,

1

2

I t

AdoRcy 7

1

pppN pN p - -

I )

AdoHat)

m G (5

mRNA guanylyltransferase

RNA(guanine-7+)methyltransferase

1

pppN p~p - - (CapO) RNA

)

I

)

RNA methyltra.nsferase

'

m G(5 ' )pppN mp~mp - - (Cap2)RNA 7

1

Figure 2. Schemattc representation of the mechantsm of cap formatton (Cap 2). Details of the scheme are given in the text. Abbreviations are pppG, GTP; N 1 and N2 , first and second nucleosides of the RNA pnmary transcript; E. mRNA guanylyltransferase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; m, methyl group.

The process of mRNA capping has been studied most extensively using vaccinia virus-derived proteins. A heterodirneric 6.5S protein complex, consisting of 90 kDa and 31 kDa subunits, was found to catalyse the reactions shown in Figure 2 (14). The 90 kDa subunit comains both the RNA triphosphatase and the RNA guanylyltransferase (the capping enzyme), while the 31 kDa subunit is required, but is not sufficient, for methyhransferase activity (15, 16). Interestingly, the capping enzyme complex is also responsible for termination of vaccinia virus transcription, although the presence of a 5 1 -cap is not a prerequisite for transcript termination (17), unlike the case of influenza virus mRNA synthesis (18) and histone mRNA 3 1 -processing (19). Vaccinia virus provides another example of a protein with effects on both 5 1 and 3 1 endprocessing; the 5 1 cap-specific viral 2 1 -0-methyltransferase also stimulates mRNA 3 1 -polyadenylation (20). Since transcript synthesis begins at the capping site, the identification of the Precise species of cap [e.g. cap A (m 7GpppAm) or cap G (m7 GpppGm)] and their adjacent sequences in RNA transcripts provided the first accurate markers of transcription stan sites on polynucleotide templates. The key to the complete 37

2: Copping and melhylolion

of mR,\'A

cbaractetrization of mRNA caps is their efficient isolation and the identification of the resulting nucleosides after cleavage of the 5' -5 ' triphosphate linkage between the m 'G and Nm. In this chapter emphasis is placed on the in vitro synthesis of highly radiolabelled capped RNA (Section 2), methods for post··transcriptional decapping and recapping of RNAs (Section 3) and the characterization of cap structures (Section 4). Methods whic h were used for discovery of the cap structure (21-23) are also described because they facilitated the isolation of capped mRNAs and the characterization of different types of caps. Readers should consult the original publications for additional details.

2. Synthesis of capped mRNAs during m vitro

transcription of viral genes 2.1 Inltroduction Several eukaryotic viruses, including mammalian reovirus, vaccinia virus and insect cytoplasmic polyhedrosis virus (CPV), contain virion-associated RNA polymerase, guanylyltransferase and methyltransferase(s). Consequently, in vitro transcription reactions mediated by purified virus particles yield capped mRNA. This has proved invaluable for determining the mechanism of viral cap formation which is essentially identical for cellular mRNAs. Since the ,·iral mRNAs, like most cellular mRNAs, initiate with either A or G, they can provide convenient sources of [3H]methyl- or 32 P-Iabelled forms of cap A (m 7GpppAm) and cap G (m 7GpppGrn) for use as authentic standard markers, e.g. in the analysis of cellular mRNA caps. Table 1 summarizes the types of cap structures that can be obtained from viral transcription reactions. Standard methods for the propaga1:ion and purification of these viruses will be found in refs 24-26.

2.2 Strategy Selective radiolabelling of specific sites in caps can be de,ised on the basi~ of the mechanism of synthesis. For example, transcription reaction mixture'

Table 1. mRNA caps synthesized in vitro by viral transcription Transcription conditions• Virus Insect c~otoplasmic polyhedros1s virus (CPVl Reovirus Vac!:inia virus

+Ado Met

-Ado Met or + AdoHey

m 7GpppA m

GpppA

m 7GpppGm

GpppG

m 7 GpppA'" m 7 GpppG'

GpppA, GpppG

• Adomet. S-adenosy1methoonone; AdoHcy, S-adenosy1homocystetne.

38

Yasuhiro Furuichi and Aaron

f. Shatkin

incubated \\ ith [ methyJ-3H] AdoMet yield I'H I methyl-labelled caps. Alternatively , [ a-31P] GTP or [ a- 3~P JA TP can be used to label either both of the 5' phosphates in m'GpppGm or only the pAm in m'GpppAm, respectively. Addition of inorganic pyrophosphate to these reaction mixtures dimirushes guanylylation of RNA and increases the proportion of ppN 5' -ends formed. In contrast, the presence of inorganic pyrophosphatase yields mostly 5' -terminal GpppN which is converted to m7 GpppNm when AdoMet is also present (Table I and Figure 2) (27). The following sections contain protocols describing the preparation of a variety of viral mRNAs bearing caps radiolabelled in different ways. For the preparation of radiolabelled m -GpppAm (or GpppA) structures, CPV would normally be the system of choice. However, this virus is usually purified from infected silkworms, a source not always readily a'ailable. Vaccinia virus also produces m7 GpppAm-ended transcripts, and it can be obtained in relatively large amounts from infected cell cultures, e.g. HeLa cells or chick embryo fibroblasts. For radiolabelled G caps (m 7 GpppGm, GpppG), reovirus mRNAs provide a good source, since milligram quantities of virions can be readily purified from a few litres of infected mouse L cells. Although the transcription and capping reactions for producing capped viral mRNAs are very similar, they have different temperature optima, etc., and the protocols described are not interchangeable. Section 4.2 contains protocols for the enzymic removal of caps from viral mRNAs for use as authentic cap markers. Non-radioacthe caps and cap analogues ha\e also been synthesized chemically and are commercially available from Pharmacia-LKB and other suppliers. The synthesis of radiolabelled capped mRNAs from cellular genes using bacteriophage promoters and RNA polymerases is described in Section 2.5. These products can be especially useful for producing large amounts of mRNA for translation studies, model pre-mRNAs for maturation studies, and capped probes for various analyses.

2.3 Synthesis of capped mRNAs using insect cytoplasmic polyhedrosis virus 3 [ H] Methyl-labelled CPV mRNA is synthesized using [ methyPH] Ado:'v1et as described in Protocol/. Note that all tubes, pipettes and solutions should be free of RNase. This can be accomplished where necessary by autoclaving or treatment with DEPC (further details will be found in Volume I, Chapter I, Section I. 1). Protocol 1. In vitro synthesis of capped CPV mRNA Equipment and reagents • S adenosyH. ·I methyi·3 H) methronine (AdoMet; Amersham, Searle; - 70 Cil mmol. 1 mCi.ml in solution in HCI, pH 2 0-2. 51; thrs should be neutralized before use by additton of an equal volume of 70 mM NH4 0H

eCPV vrrrons (1 -2 mg 'ml, prepared by standard methods described in ref. 24 • 1 .0 M Trrs-HCI. pH 8.0 e0.1 M MgCI2

Continued 39

2: Capping and methylation of mRNA Protocol 1. Continued • Stock solutions 140 mM) of ATP, CTP, GTP. and UTP prepared m H2 0 , neutralized to pH - 7 with Tns base, and stored frozen • Eluuon buffer (50 mM Tris-HCI , pH 8 .0)

e Sephadex G- 100 column 120cm x 0.6 em) equilibrated in elution buffer • Phenol. chloroform ( 1: 1. vlvl • 3 .0 M potass1um acetate, pH 4. 5 e Ory

ICe

Method 1. Set up the transcription reaction by mixing the following reagents in a sterile microcentrifuge tube on ice: e 1.0 M Tris-HCI, pH 8.0 5 1-11 e 0. 1 M MgCI 2 e 40mM ATP e 40mM CTP e 40mM GTP

12 1-11 101-11 51JI 51-11

e 40mM UTP 51-11 • ( methyi-3 H] Ado Met (20 iJCi) 40 1-11 • CPV virions ( 10-20 IJg) 1- 21-11 to 100 IJI final volume • H 20 2 . Incubate the mixture at 31 °C for 2 h. 3 . Add an equal volume (100 IJI) of phenol :chl·oroform ( 1: 1) and vortex for 1 min . 4 . Centrifuge the mixture at 10 000 g for 5 min separate the phases.

1n

a microcentrifuge to

5 . Carefully remove the upper (aqueous) phase to a clean microcentrifuge tube . 6. Separate the radiolabelled CPV mRNAs 1'rom the unincorporated ( methyi-3 H) Ado Met by chromatography on Sephadex G- 100. Collect 0 .1 ml fractions and monitor the separation by scintillation counting. 7 . Pool the fractions containing the CPV mRNAs (first peak) in microcentrifuge tubes and add 3M potassium acetate. pH 4 . 5, to 0 . 15 M final concentration plus, 2. 5 vol. of ethanol. Place 1:>n dry ice for 15 min to precipitate the RNA. and then pellet the precipitated RNA by centrifugation in a microcentrifuge tor 1 5 min. B. Store the RNA as a wet pellet at - 20 ° C or dry it and redissolve it in autoclaved or DEPC-treated, nuclease-free dis.tilled H2 0 before freezing.

Under the conditions described in Protocol I about 50Jo of the [ 3H] met h) I groups are incorporated into m7GpppAm from the [methyPH]Ado~le( precursor. To obtain [ 3H]methyl-labelled m' GpppAm, the mRNA products are digested with nuclease PI, and the caps are purified by high-\oltage paper electrophoresis or chromatography on polyethylenimine (PEr) cellulose or paper (see Section 4). 40

Yasuhiro Furuichi and Aaron]. Shatkin To prepare 32 P-Iabelled unmethylated GpppA, carry out the RNA synthesis in a modified transcription reaction mixture (Protocol I, step I) containing 2 mM S-adenosylhomocysteine (Ado Hey) in place of [ methyPH] AdoMet and with t he addition of 0.4mM [f3-32P]ATP in place of the ATP. Prepare the [ f3-3 2P] A TP from h-32 P] A TP according to the method of Furuichi and Shatk.in (28).

2.4 Synthesis of capped mRNAs using reovirus Protocol 2 describes the preparation of washed reovirus cores and their subsequent use in an in viuo transcription system with [ methyJ-3H] AdoMet and I a-32 P l GTP. Protocol 2 . Synthesis of capped mRNAs using washed reovirus cores Equipment and reagents • Punfied reovirus (- 5 mg/ml) prepared by standard methods as described in ref 25

e V1ral core washing buffer (50 mM TrosHCI, pH 8.0 50 mM KCII

e 10 mg/ml a-chymotrypsin (Worthington Biochemical, crystalline enzyme freshly dissolved m H 2 0)

• 0 . 25M phosphoenolpyruvate

• 1 mg ml

• [ methyi-3 H JAdoMet, 1 M Tris-HCI, pH 8 0 , 40 mM stock solutions of ATP, GTP, CTP, and UTP, phenol· chloroform, elution buffer, and Sephadex G-1 00 column as described in Protocol 1

inorganic pyrophosphatase (Boehringer Mannheim, 1 mg/mll

e 3.0 M KCI • 1.0 M magnes1um acetate, pH 7.0 • [a-32P]GTP (Amersham , Searle; 410 Cil mmol, 10 mCi/ml)

• Pyruvate kinase (Calb1ochem, - 8450 IU/ml )

Method 1. Using stock solutions, prepare reovirus cores by mixing 2 mg (measured as protein ) of purified reovirus in buffer consisting of 70 mM Tris- HCI, pH 8, and 90 mM KCI (final concentrations). Add 10 mg ml chymotrypsin to 0.5 mg/ml and mcubate the mixture (total volume of 2 ml) for 45 min at 42 °C.

2 . Collect the viral cores by centrifugation at 10 000 g for 10 min at 4 °C 3 . Resuspend the pellet in - 0 . 5 ml of washmg buffer and centrifuge again as m step 2. Repeat. 4 . Resuspend the washed viral cores in 0 2 ml of washing buffer. 5. Set up the transcription react1on by m1xmg the following reagents in a m1crocentnfuge tube on 1ce:

• 1.0 M Tris-HCI. pH 8 .0 • 1.0 M magnesium acetate, pH 7.0 e 40mM ATP Continued

41

2. Copping and methylation of mRl\'A

Protocol 2 . Continued 50~o~l

e40mM CTP e 40mM UTP

50~o~l

e40 mM GTP

50 ~o~l

e [cx-32P]GTP (1001JCi)

101JI

•

[methyi-3 H]AdoMet

100~o~l

(501JCi)

• 0. 25 M phosphoenolypyruvate

10 iJI

• pyruvate kinase (- 8 units)

1 IJI

• washed reov1rus cores (equivalent to 500 IJg v1rus measured as protem) • 1 mg/ml inorganic pyrophosphatase

50 1-11 1 1-11

• H2 0 to 0. 5 ml final volume 6 . Incubate the mixture at 45 °C for 2 h with shaking every 1 5 min to resuspend cores. 7 . Recover and store the capped, radiolabelled reovirus mRNAs as described in Protocol 7, steps 3-8 except in step 6 collect 0. 5 ml fractions from the Sephadex column.

To obtain the [ 3H] methyl- and 32P-labelled m-GpppGm caps, the reoviru transcripts are digested with nuclease PI and the caps are isolated as described in Section 4.

2.5 Synthesis of capped mRNAs using vaccinia virus In vitro transcription using purified vaccinia virus yields poly(A)- mRN -\ containing m 7GpppAm and m7GpppGm in a 2: I ratio (29). These two cap forrr' may be released from the transcripts by nuclease PI digestion and are read1 resolved by paper chromatography (see Section 4.3.2). The transcriptK 1 procedure is described in Protocol 3.

Protocol 3 . In vitro synthesis of capped vaccinia virus poly(AJ mRNAs

Equipment and reagents e vacc1n1a virions (2-4 mg/ml calculate from the A 260 value where 1 mg-ml is eqUivalent to - 15.7 A 280 units) punfied by standard methods as described 1n ref 26 e 1% fvtv) Nonidet P-40 INP-40· GIBCO-

BRLI

e o.2 M DTT (Calbiocheml e 1.0 M Tris-HCI, pH 8.0 0 . 1 M MgCI, 40 mM stock solutions of A TP. CTP GTP and UTP. (methyi-3 H )AdoMet phenol:chlorofor;n, elution buffer Sephadex G-100 column• as described m Protocol 1

Continued 42

't'asuhiro Furuichi and Aaron /. Shatkin Protocol 3 . Contmued Method 1. Set up the transcnption reaction by m1xing the follow1ng reagents microcentrifuge tube on ice: e 1.0 M Tris-HCI, p H 8.0

10 IJI

e 0.1 M MgCI 2

101JI

e 40 mM A TP

1 2 Ill

e 40mM CTP

1 21JI

e 40mM GTP

1 21JI

e 4 0mM UTP

1n

a

1n

a

51JI

e 1% NP-40

10 Ill

e 0.2M OTT

10 IJI 401JI

• [methyi-3 H]AdoM et (201JCi) • Purified vaccinia virus ( 100 IJg) e H 20

501JI t o 200 IJI final volume

2 . Incubate the mixture at 37 °C for 2 h. 3 . Centrifuge the m1xture at 10 000 g for 1 5 min at room temperature microcentnfuge to pellet the vtrions.

4 . Carefully remove the supernatant to a fresh m1crocentrifuge tube and purify the viral mRNAs as described in Protocol 1 steps 3 - 8•. • Since the vaccm1a v1rus mRNAs are polyadenylated, standard aHm1ty chroma to· graphy on oligo(dTI - cellulose described in ref. 30 may be used for their purification in place of Sephadex G- 100 chromatography.

2.6 In vitro transcription and capping of cellular mRNAs using bacteriophage RNA polymerases In vitro transcription systems based on SP6, T3 or T7 phage promoters and RNA polymerases make it possible to synthesize biochemical amounts of RNA molecules of any desired sequence including cellular mRNAs (31 ). The transcripts have been widely used not only for expression cloning but also for the investigation of RNA processing, RNA stability, and protein synthesis. Although bacteriophage polymerases transcribe the DNA sequences placed downstream of their specific promoters very accurately and efficiently, the 5 ' -ends of the Products are not capped and remain triphosphorylated. However, for some purposes, such as studies on RNA splicing and/ or translation, it is important to use capped transcripts. By including vaccinia \irus-derived capping enzyme and AdoMet in the transcription incubation mixture, the 5 -triphosphorylated RNA can be readily capped and methylated according to the reaction sequence depicted in Figure 2. Alternatively, cap analogues such as GpppG or m 7GpppG can be added to the transcription mixture. By concomitantly decreasing the GTP concentration, transcription by the phage polymerase then starts prefe rentially 43

2: Capping and methylation of mRl\'A

with GpppG or m 7GpppG. Chain elongation is still effective at the lower GTP concentration. A procedure for the synthesis of capped mRNA using SP6 polymerase is described in Protocol 4. Protocol 4. Synthesis of capped cellular mRNAs using SP6 RNA polymerase Equipment and reagents e 1 0 M Tris-HCI, pH 7.5

• RNasin (Promega, 20 units 'IJI)

• 0 . 1 M MgCI2 , Sephadex G· 100 column, elutton buffer. phenol:chloroform as described in Protocol 1

• [ar-32P) UTP (Amersham, Searle; 400Ci•mmol. 10mCi/ml)

• 20 mM spermidine-HCI

• 50 mM stock solutions of A TP, CTP, and UTP

e 2 mg/ml BSA

e 5mM GTP

• SP6 RNA polymerase (Promega, 1 0-20 UnttS/IJI)

• 50 mM m'GpppG (Pharmacia-LKB)

• Template DNA. lineanzed. 1 mg ml • 0 1 M OTT !Calbiochem)

• RNase-free DNase (Promega, 1 U IJI, 0 .21JQ 'IJI)

Method 1 . Set up the transcription reaction by mixing in a microcentrifuge tube (at room temperature to avoid precipitation of the DNA by spermidine): • DNA template • 1.0 M Tris-HCI, pH 7. 5 e o . 1M MgCI 2 e 0.1 M OTT

10 111 41JI 61JI 10111

• 2mg/ml BSA

51JI

e 50mM ATP

1 Ill

e 50mM CTP

1 Ill , Ill

e 50mM UTP e 5mM GTP

, Ill

e [a-32P]UTP (201JCil

21-11

e 50 mM m 7GpppG

1 Ill

• RNasin • 20 mM spermtdine-HCI • SP6 RNA polymerase • H2 0

51JI 10 Ill 41JI to 100 1-11 final volume

2. Incubate the mtxture at 40 °C for 1 h.

3. Remove the template DNA by adding 2 IJI RNase-free DNase (final cone 20 IJg/ml) and incubate at 37 °C for 10 mtn. 4. Recover the capped transcripts as described in Protocol 1, steps 3-8

44

Yosuhiro Furuichi and Aaron ] . Shotkin

3. Site-specific radiolabelling of caps in

cellular mRNAs 3.1 Introduction Characterization of the cap structures in indtvidual cellular mRNAs is often a difficult task because of the limited amounts of material available. Hence, it is important to be able to label the cap and/ or the adjacent nucleotide specificaiJy and to a high specific radioactivity in order to establish the 5 1 -terminal structure of mRNAs. This can be accomplished by one of the following three approaches: e labelling with 32 P by decapping and recapping (Section 3.2) elabelling with 3H by periodate oxidation and borohydride reduction (Section 3.3) e labelling with 3H by 2 1 -0-methylation (Section 3.4) The first approach, radiolabelling of mRNA by chemical removal of the m7G followed by enzymatic recapping in the presence of [a-32P]GTP, is probably the most practical procedure since purified guanylyltransferase (capping enzyme) is now commerically available (Gibco-BRL). In addition, purification procedures for guanylyltransferases from rat liver (32), yeast (Saccharomyces cerevisiae) (33, 34) and brine shrimp (Anemia salina) (35) have been described. These enzymes can also be used for recapping. Radiolabelling by oxidation and 3H-borohydride reduction, the second approach , has the disadvantage that the 5 1 -terminal m 7 0 is converted to the trialcohol derivative in this procedure. In addition, because the 3 '-terminal residue in RNA also contains a free cis-dioi, it becomes 3H-labeliled by the oxidation and reduction scheme. The third technique, labelling with 3H by 2' -0-methylation, requires that the mRNA contain a Cap 0 structure, a limitation because most animal virus and ceUular mRNAs contain Cap I or 2 structures.

3.2 Radiolabelling mRNAs by decapping and recapping This labelling strategy comprises the following steps: • chemical removal (decapping) of the 5 -terminal m70 from capped mRNA • recapping with guanylylt ransferase and [ a-32p] GTP I

3.2.1 Decapping mRNA Caps contain a free 2 -3' cis-dioi on the ribose ring of the 5 -linked m7 G. I

I

These hydroxyl groups can be oxidized to the dialdehyde form by treatment With sodium metaperiodate (Na104 ) under conditions that leave the RNA chain intact (with the exception of the 3 1 -terminal nucleotide which also contains an oxidizable cis-diol). The decapping procedure, described in Protocol 5, 45

2: Capping and methylation of mRNA

exploits this periodate oxidation followed by a ~-elimination reaction with aniline. The oxidation and ~-elimination reactions convert the mRNA 5 ' -caps to 5 ' -triphosphorylated ends with release of the trialcohol derivati'e of m7G. referred to as m ~G+ :

Protocol 5 . Chemical decapping by oxidation and ~-elimination reaction

Reagents • Capped cellular mRNA I- 100 IJg/mll purified as descnbed in Volume I. Chapter 1. Section 3 • Metapenodate reagent (freshly prepared, 10 mM Nal04 1n 0.15 M sodium acetate, pH 5. 3) • 0 . 3 M aniline, pH 5.3

• 20% glycerol • 2 .0 M ammonium acetate, pH - 7 .0

e TSE buffer 120 mM Tris -HCI. pH 7.4, 0. 5mM EDTA, 0 .5% SDSI • Carner yeast tRNA !S1gmal dissolved in H 2 0 at 2 mg ml

Method Periodate oxidation 1. Mix the capped cellular mANA (- 1 IJg) with 20 IJg of carrier tRNA in 10 IJI H2 0. 2. Add 1 IJI of periodate reagent and incubate the mixture on ice for 1 h in the dark. 3 . Add 10 1-11 of 20% glycerol to stop the oxidation.

4 . Precipitate the RNA by adding 0.15 ml of 2.0 M ammonaum acetate, pH - 7 .0 and 0 . 85 ml of cold (- 20 °C) ethanol. 5 . Mix well and leave at - 70 °C for 1 5 min. 6. Centrifuge the tube at 10 000 g for 15 min at 4 ° C. Carefully remove and discard the supernatant. 7 . Redissolve the RNA pellet in 0 . 18 ml TSE buffer. Add 0 .1 8 ml of 2.0 M ammonium acetate, pH -7.0, followed by 0 .9 ml ethanol. 8. Repeat steps 6 and 7. 9 . Dry the RNA pellet under reduced pressure and redissolve it in 20 IJI of TSE buffer.

/3-elimination reaction 10. Add 0 . 1 ml of 0.3 M aniline, pH 5.3 , mix and incubate the mixture for 5 min at 30 ° C .

Continued 46

Yasuhiro Furuichi and Aaron f. Shatkin Protoc o l 5 . Continued

11 . Precipitate the RNA by adding 0.1 ml of 2 0 M ammonium acetate, pH -7.0, and 0.55 ml of cold (- 20 °C) ethanol.

12. Recover the RNA as descnbed m steps 5 -8. 13. Redissolve the decapped RNA in 101-11 of distilled H 20

3.2.2 Recapping mRNA The decapped triphosphorylated RNA is recapped by incubation with the vaccinia vi rus capping enzyme complex which contains RNA triphosphatase and methyltransferase(s) as described in Prorocol 6. The reaction sequence is: pppNm- __. ppNmwhere

* and

la-J~P]GTP

*

GpppNm-

[meth~PHIAdo\1 et,

*

m'GpppNm-

• denote 3 ~P- and [3H] methyl-Iab,elled positions, respectively.

Protocol 6 . Recapping mRNA with [a- 32 P jGTP

Reagents • 2 mM S -adenosylmethtontne IAdoMet, Boehnnger Mannhetm) dissolved in dilute HCI at pH - 2 and stored frozen; just before use neutralize w1th an equal volume of 70 mM NH4 0H

• Decapped mANA 1- 0. 5-1.0 IJ9 tn - 5-1 0 IJI H ~Ol prepared as in Protocol 5• e 1.0 M Tris-HCI, pH 7.9 • 12 mM MgCI ~ e 0 . 15M KCI

• Phenol:chloroform (1: 1, v'vl. Sephadex G-1 00 column, elut1on buffer, and 3.0 M potasstum acetate, pH 4 . 5 as in Protocol 1

• 0.125 M OTT (Calbiochem) • 500 mg/ml BSA • Guanylyltransferase from vacctnia vtrus (Gibco-BRL catalogue # 8024SA 1-5 U 'IJI)

e 0.25 M phosphoenolpyruvate, pyruvate kinase. 1a- 32P GTP as 1n Protocol 2

Method 1 · Set up the recapping reaction by m1xing ttle following reagents in a microcentrifuge tube on ice: • 1.0 M Tris-HCI, pH 7.9 • • • •

2.51JI

12 mM MgCI 2 0. 1 5 M KCI 0.125 M OTT 500 mg/ml BSA

5 IJI 2 Ill 11JI 5 1-11

• [a-32P] GTP ( 100 IJCil

10 Ill

Continued 47

2: Copping and methylation of mRNA Protocol 6 . Continued • 2 mM AdoMet

51JI

• decapped RNA (0. 5-1 .0 IJQ)

10 Ill

• guanylyltransferase (1-5 umts) 1 IJI to 50 1JI final volume • H2 0 2 . Incubate the mixture for 60 min at 37 °C. One unit of guanylyltransferase will form - 1 pmol of cap 1n 30 m1n under these cond1t1ons. 3 . Add an equal volume of phenol:chloroform ( 1: 1) and vortex for 1 min. 4. Recover the RNA by ethanol precipitation as described 1n Protocol 1, steps

4-7. 5 . Rinse the RNA pellet w1th 50 IJI of 75% ethanol at - 20 ° C. 6 . Dry the pellet and redissolve the rad1olabelled RNA in 10-20 IJI of H 20 . 7 . Store the RNA at - 70 °C or in liquid nitrogen. • The decapped mANA will also contain earner tRNA .

After the recapping procedure, the recapped RNA may be subjected to cap analysis (Section 4) to document the presence of m7GpppNm. Figures 3 and 4 show typical cap analyses for a \ariety of mRNAs. Unmethylated caps of the structure GpppNm are formed if the recapping (see Prorocol 6) is carried out either in the absence of AdoMet or in the presence of 2 mM AdoHcy.

3

2 :+

c;ppp .\

m

4

5

6

·* ,\ m ur <.ppp

• I

- - ..... • no RNA

CPV mRNA

ppG-RNA

EIA mRNA
~

- orlrtn

ppG-RNA EIA t ranscript

Yasuhiro Furuichi and Aaron ]. Shatkin

A

2

3

4

EB

8

8

5 Pi

-- · none TMV globin CPV

-

m 7G pppm 6 \m

-

rn GpppA

1

01

· - - • -ori gin

2 3

· J5

Figure 4 . Analys1s of decapped and recapped 5'-termln! by DE81 paper electrophoresis and thtn layer chromatography on PEl -cellulose. (A) Decapped RNAs were recapped by 1ncubat1on With [a-32P]GTP 1n the presence of AdoMet (see Protocol 6). digested With nuclease Pl (see Protocol9), and analysed by electrophoresis on DE81 paper (see Protocol16). Lane 1, no mANA; lane 2. tobacco mosa1c virus (TMVl RNA , 1 l)g; lane 3, rabbit glob1n mANA, 0.1 l)g: lane 4, CPV mANA, 0 . 5~Jg ; lane 5, ch1cken US snRNA 0.41)g (B) ldent1f1ca110n of m 1GpppA"' and 7 m Gpppm6A'" cap structures by thin-layer chromatography on PEl-cellulose. A her electro· phoresis as shown tn (A). 32P-Iabelled cap structures were eluted from the DE81 paper in 1 M triethylammonium bicarbonate, pH 7.6. and further analysed by chromatography on PEl-cellulose (see Protocol 75) the developing solvent was 0.6 M LiCI. Lane 1 m 7Gpppm6A"' of globin mANA (Upper spot from lane 3Al; lane 2, m 7GpppA"' CPV mANA cap (upper spot from lane 4Al; lane 3, m7GpppA of US snRNA (upper spot from lane SA). (Data ktndly prov1ded by Or K Mizumoto.)

Figure 3. Rad1olabelhng of 5 -caps tn viral mRNAs and analys1s by DE81 paper electrophoreSIS, CPV mANA and adenov1rus E1A mRNAs expressed tn HeLa cells and yeast respectively were decapped and recapped with [a- 32P]GTP as descnbed 1n Protocols 4-6. After d1ges110n w ith nuclease P1 (see Protocol 9), the caps were analysed by electrophoresis on DE81 Paper (see Protocol 16) Lane 1, no mANA; lane 2, decapped and recapped CPV mANA, lane 3 , CPV RNA (negative control, synthes1zed in the presence of AdoHcy and subjected to all the same steps as CPV mANA and, 1n add1t1on , alkaline phosphatase treatment to remove the unprotected 5'-dlphosphate); lanes 4 and 5, adenovirus E1A mRNAs (transcription starts With Bither A or G); lane 6, CPV RNA (positive control, synthesized in the presence of AdoHcy and thus conta1ning ppG 5'-ends. capped in a modified capping reacuon with la.-32P]GTP but w1thout Adometl. Pos1t1ons labelled with ' 2P are Indicated by * . Data kmdly provided by Dr Kiyoh1sa Mizumoto (Kitasato Un1vers1ty, Tokyo, Japan).

49

2: Capping an d methylation of m RNA

3. 3 Labelling of mRNA by period ate oxidation followed by

reduction with [ 3H] sodium borohydride As mentioned in Section 3.2.1, the free 2 ' -3 ' cis-diol on the ribose ring of the 5 ' -linked m 70 can be oxidized to the dialdehyde form by treatment with sodium metaperiodate (Na10 4). The dialdehyde can then be radiolabelled by reduction to the dialcohol form (m7G ' ) with [ 3H] NaBH 4 according to the following scheme where * indicates radiolabelled positions: Nal04

m7GpppNm---+ m7 GpppNm-

DoH

[l H ]NaBH 4

~cHo

m 7G ' pppNm -

c~~H20H

This labelling procedure is described in Protocol 7.

Protocol 7 . Labell ing of m ANA by reduction wit h [ 3 H ]NaBH 4 Equipment and reagen ts • Capped mR NA ( - 100 llg /ml) isolated as in Volume I, Chapter 1, Protocol 4 • 20 mM sodium acetate buffer. pH 5.0 • 10% propylene glycol

e o.3 M sodium acetat e buffer, pH 5.0 • 0. 5 M sodium phosphate buffer,

•

[

3

H] NaBH 4

(New

England

Nuclear;

8.8 Ci/mmol, 10 mCi/ 10 Ill in 50 mM NaOH) • Elution buffer, Sephadex G- 100 column , and 3.0 M pot assium acetate. pH 4.5, as in Protocol 1 and metaperiodate reagent as in Protocol 5

pH 7 .0

Method 1 . Dissolve 2- 51Jg of RNA in 0.1 ml of 20 mM sodium acetate buffer, pH 5.0. 2 . Add 10 Ill of metaperiodate reagent and incubate the mixture for 1 h at 0 °C in the dark. 3 . Destroy the excess Nal04 by adding 20 Ill of 1 0% propylene glycol and incubate for a further 10 min as in step 2.

4. Add 0 . 15 ml of 0.3 M sodium acetate buffer, pH 5.0, and 0.6 ml of cold (- 20 °C) ethanol. 5 . Recover the oxidized RNA as described in Protocol 5, steps 5 and 6.

6 . To remove propylene aldehyde, redissolve thE! RNA pellet in 1 50 Ill of H 2 0 and reprecipitate it as in steps 4 and 5.

Continued 50

Yasuhiro Furuichi and Aaron ]. Shatkin Protocol 7. Continued 7 . Wash the RNA pellet with ether, air dry it and redissolve it in 30 IJI of 0.5 M sodium phosphate buffer, pH 7.0 .

8 . Add 21JI of [ 3 H] NaBH 4 (2 mCi) and incubate the mixture on 1ce for 1 h. 9 . Remove unincorporated [ 3 H ] NaBH 4 by chromatography on Sephadex G-1 00 and then recover the radiolabelled RNA by ethanol precipitation as descnbed in Protocol 1, steps 6 and 7.

10. Store the labelled RNA frozen either as an ethanol pellet or dried and dissolved in RNase-free distilled H2 0 (see Protocol 1, step 8).

3.4 Labelling of mRNA by 2'-0-methylation 2 ' -0-Methyltransferase catalyses the transfer of the methyl group from AdoMet to the 2' -OH of the nucleotide immediately adjacent to the cap nucleotide in capped mRNAs: AdoMet

m7GpppNm- _____. m 7GpppNm-

The 2 ' -0-methyltransferase has been purified from vaccinia virus and used for radiolabelling the R NAs of brome mosiJ.ic virus and tobacco mosaic virus by conversion of 5' -terminal m7GpppG to m 7GpppGm (36). For efficient methyl group transfer, vaccinia virus 2' -0-methyltransferase requires a polyribonucleotide substrate capped with a Cap 0 (m7 GpppN) structure. Since mRNAs from lower eukaryotes and plants contain mainly Cap 0 structures, they are good substrates for the enzyme. The procedure for labelling mRNA by 2' -0-methylation with [ methyPH] AdoMet is described in Protocol B.

Protocol 8. Labelling of mRNA by 2'-0-methylation Equipmen t and reagents • 1.0 M Tris-HCI, pH 7. 6 • RNA contatn~ng m 7 GpppN (Cap 0) structures ( 1 mgl mll • [ methyi-3H] Adomet. phenol:chloroform,

elution buffer, 3.0 M potassium acetate, pH 4.5, and Sephadex G-100 column as in Protocol 1; 0 . 125 M DTI and vaccinia capping enzyme (guanylyltransferase) as in Protocol 6

Continued

51

2: Capping and methylation of mRNA

Prot ocol 8 . Continued Method 1. Set up the methyltransferase reaction by m1x1ng in a microcentnfuge tube. 5 ~I e 0 . 125M OTT 10~1 e [methyi-3 H )AdoMet (20 !JCI) 40 !JI • m 7GpppN RNA 10 !JI 2 ~I • vaccm1a capping enzyme (2-1 0 units) • H2 0 to 0. 1 ml final volume 2. Incubate the mixture at 37 ° C for 30 min. 3 . Purify the radiolabelled RNA as described in Protocol 1, steps 3- 8 . • 1.0 M Tns-HCI, pH 7 .6

4. Characterization of caps 4.1 Strategy The first stage in the analysis of cap structures in mRNA is the enzymic removal of the cap from the mRNA chain. The m7GpppN and GpppN are resistant to various nucleases that hydrolyse phosphodiester bonds in DNA and RNA. In addition, 2' -0-methylation of the ribose moiety in an oligonucleotide renders the 3 -5 ' phosphodiester linkage adjacent to the cap resistant to RNases that cleave RNA via 2 ' -3' phosphate cyclization. Consequently, Cap I and Cap 2 structures (i.e. m7GpppNmpNp and m7GpppNmpNmpNp) that are common in mRNAs of higher eukaryotic cells, are resistant to RNase T2, a nuclease that hydrolyses RNA without base specificity. Nuclease PI will also remove m7GpppNm from mRNA, but because nuclease PI hydrolyses RNA to 5 -mononucleotides it is unaffected by 2 ' -0-methylation. In addition, due to its mechanism of phosphodiester cleavage, nuclease PI digestion of capped mRNA yields m7GpppNm, i.e. caps without a ] ' -phosphate group. A variety of separation methods are available for the analysis of the cap structures released by enzymic digestion. These include various forms ol chromatography and electrophoresis, as well as two-dimensional procedures. Because purified mRNA is usually available in onJy small amounts, the recapping method described in Section 3.2.2 is recommended for cap analys1s. Thb approach can provide highly 32P-labelled mRNA, since the reaction takes plan : specifically at the 5' -terminus and is not usually accompanied by ~puriou' radiolabelhng (37). Concerning the separation of radiolabelled caps, the combination of one-dimensional paper electrophoresis (Section 4.3.4) and paper chromatography (Section 4.3.2) of nuclease PI digested RNA, as shown in Figure 6, would suffice for the identification of cap species. The specific cleavage of the cap 5 -5 ' pyrophosphate linkage and analym of the resulting products is necessary for confir mation of structure and further I

I

I

52

Yasuhiro Furuichi and Aaron}. Shatkin characterization of the nucleoside N<mJ following the m7 G. Several enzymes are known which can cleave the cap pyrophosphate linkage, including the nucleotide pyrophosphatases from snake venom (22), tobacco (38), potato (39) and yeast (40) as well as a m7G-specific pyrophosphatase from Hela cells (41, 42). The choice of enzyme is usually dictated by both purpose and availability. Some enymes may also be used to prepare RNA chains free of caps as indicated below. (a) Snake venom nucleotide pyrophosphatase catalyses the con\ersion of m7GpppNm to m~Gp + PiT pNm. It is often used for the complete djgestion of caps (17). However, because it is always contaminated with traces of phosphodiesterase, snake venom pyrophosphatase cannot be used to obtain 'decapped' RNA chains. (b) Nucleotide pyrophosphatases from tobacco tissue culture cells (38) and

potato (39) are, in contrast, nuclease-free which also makes them convenient enzymes for removing the cap from mRNA without damaging the polynucleotide strand: m7GpppNm - - - + m7Gp + Pi + pNmDigestion with tobacco pyrophosphatase (followed by phosphatase treatment) is particularly valuable for preparing the 5 ' -end of mRNA for subsequent radiolabelling by incubation with polynucleotide kinase and ['y-32 P) A TP (see Volume I, Chapter 2, Protocol 2). (c) m 7G-specific pyrophosphatase from HeLa cells recognizes the m70 in caps and cleaves the pyrophosphate linkage in capped short oligomers (41). This enzyme, m 7 0-specific Hela nucleotide pyrophosphatase, catalyses the following reaction: m7GpppNmpNp-- m7Gp + ppN"'pNpThe enzyme preferentially cleaves the pyrophosphate linkage adjacent to the m70 residue and leaves a 5 ' -diphosphate on the residual oligonucleotide. Caps containing trimethyl G derived from U2 snRNA and unmethylated GpppN structures are not substrates for the Hela pyrophosphatase (42). Unlike tobacco p}"rophosphatase, the HeLa enzyme apparently does not attack m'G-capped polyribonucleotides that are longer than about 10 residues. Tbjs enzyme is present mainly in the cytoplasmic fraction of HeLa cell extracts. In the intact cell, it presumably scavenges capped oligonucleotides resulting from mRNA turnover that might othemise inhibit the initiation of translation. (d) Yeast mRNA-decapping enzyme is a pyrophosphatru.e that cleaves m-GpppN and GpppN but not pppN end groups in RNA; it has been partially purified from yeast (40). The nuclease-free preparation catalyses the release of m 'Gpp and Gpp from mRNA and capped synthetic polyribonucleotides 53

2 Capping and methylation of mRSA at rates directly dependent on chain length. For instance, a 540mer was a 10-fold better substrate than a 50mer. Predictably, pretreatment with RNase A or nuclease PI renders capped RNAs inactive as substrates. The following now chart illustrates two different pathways for analysis of caps by enzymatic digestion. Both pathways permit the purification of nuclease-resistant 5 ' -terminal caps from the vast numbers of internal ribonucleotides which then simplifies the subsequent analysis of cap constituents. Capped mRNA

Nuclease PI m-GpppX' +pY'"+pZ+ Pi

l

Teoba~co p~

ropho>phata>e

,T·,z,

"""':~G:: ...

SnaJ.e 'cnom p} ropho>phatase

pXm 1 pYm I pZ + Pi

Note that initial cleavage with RNase T2 retains the methylated nucleoside' (Xm, ym) and another residue (Z) in phosphodiester linkage as part of a

extende-d cap structure. By contrast. nuclease PI treatment releases the ca, and y m and Z as 5' -mononucleotides; nuclease PI also has a 3' -phosphatase activity. The constituent nucleotides in the capped oligonucleotide can be identified at various stages b:y two-dimensional thin-layer cellulos~ chromatography (43).

4.2 E.nzymic analysis of caps 4.2 .1 Removal of caps from mRNA Protocol 9 describes the removal of caps from mR As asing either nuclease: PI or J~Nase T2. This procedure may also be employed for preparing radiolabelled cap structures for use as markers for analytical purposes, e.g. see Figures 5 and 6 (Section 4.3). 54

Yasuhiro Furuichi and Aaron /. Shotkin Protocol 9 . Removal of caps with nuclease P1 or RNase T2 Reagents • Aad1olabelled capped mANA (- 1-2 mgl ml) prepared as m Protocols 1-4.

• ANase T2 !Calb1ochem) • 0.1 M sod1um acetate buffer. pH 6 0

6 -8

e 0.1 M sod1um acetate buffer, pH 4 . 5, contam1ng 10 mM EDTA

• Nuclease P1 !Calb10cheml

Method A. D1gestion with nuclease P1 1. Dilute the capped RNA to a final concentration of -0.5 mg/ml and 10 mM sodium acetate buffer. pH 6.0. using the 0.1 M stock solution of 0.1 M sodium acetate, pH 6.0 . 2. Add nuclease P 1 to 50 JJg/ml. 3. Incubate the mixture at 37 °C for 30 min. 4 . Proceed to Protocol 16 (or another purification or analytical technique)

B. Digestion With RNase T2 1. Dilute the capped mANA to a fmal concentration of - 0. 5 mg ml in 0 1 ml of 10 mM sodium acetate, pH 4. 5, and 1 mM EDTA using the 0.1 M stock solution of 0 . 1 M sod1um acetate, pH 4. 5, 10 mM EDTA. 2 . Add RNase T2 to 50 U ml final concentration. 3 . Incubate the mixture at 37 °C for 2 h. 4 . Proceed to Protocol 12 (or another purification or analytical method).

4.2.2 Enzymatic cleavage of caps with nucleotide pyrophosph atases Protocols 10 and II described the cleavage of caps with snake venom pyro-

phosphatase and tobacco pyrophosphatase, respectively. Protocol 10 . Cleavage of caps with snake venom pyrophosphatase to identify the Nm in m 7GpppNm Reagents • m 7GpppN'" caps removed from capped mANA by nuclease P1 digest1on !see Protocol 91 and isolated by paper electro· phores1s as 1n Protocol 14 (Secuon 4.3 21. see also Figure 68

• Snake venom nucleotide pyrophosphatase (Sigma) dissolved m H 20 at - 1 Ul ml e 1 0 M Tris-HCI, pH 7 5 e 10 mM MgCI,

Method 1. Dissolve the caps 1n 8 JJI of 20 mM Tris-HCI, pH 7. 5, and add 1 JJ) of 10 mM MgC12 and 1 JJI of nucleotide pyrophosphatase

Continued 55

2: Capping and methylation of mRNA Protocol 10 . Continued 2. Incubate the mixture at 37 °C for 30 min. 3. Freeze and lyophilize the sample for subsequent analysis by high-voltage paper electrophoresis (see Protocol 16) or two-dimensional chromatography (see Protocol 7 7 and Figure 7).

Treatment of capped mRNA with tobacco pyrophosphatase is used to prepare uncapped mRNA (containing a 5 '-terminal pNm) by releasing the cap constituents as m7Gp and Pi. After removal of the 5 '-monophosphate by phosphatase treatment, the RNA can then be 5 ' end-labelled by incubation with polynucleotide kinase and [ y-32 P] ATP (Volume I, Chapter 2, Protocol 2). Protocol 1 1. Removal of caps from m ANA with tobacco pyrophosphatase

Equipment and reagents one of the methods described tn Volume I, Chapter 1, Section 3

• 0.25 M sodtum acetate buffer, pH 6.0

e o. 125M on • Tobacco actd pyrophosphatase (Sigma, -1000U/mll • Capped mRNA (1 mgl mll isolated by

• Phenol:chloroform, elution buffer, Sephadex G-1 00 column, and 3 M potassium acetate, pH 4.5, as described in Protocol 1

Method 1. Mix in a microcentrifuge tube: • 0.25 M sodium acetate buffer

1 (JI

e 0.125M OTT

11JI

• tobacco acid pyrophosphatase

1 (JI

• capped mANA

1 (JI

• H2 0

to 10 (JI final volume

2. Incubate the mixture at 37 °C for 1 h. Stop the reaction by placing the tube on ice. 3 . Spot an aliquot of the reaction mixture on paper and analyse it according to Protocol 1 7, (Section 4 . 3. 5; see also Figure 7) for confirmation of the release of m 7 Gp (identified by scintillation counting if radiolabelled or by fluorescence under UV light if present in sufficient amount).

4. Purify the decapped RNA according to Protocol 1, steps 3 - 8.

The decapped RNA can be 32P-Iabelled at the 5' -end by first removing the residual phosphate by phosphatase treatment (e.g. with I0 units of calf intestinal alkaline phosphatase per millilitre in 50 mM Tris-HCI, pH 8.0, for 60 min at 37 °C} and then incubating the repurified RNA with polynucleotide kinase and [y-32P]ATP (44). 56

Yasuhiro Furuichi and Aaron ]. Shatkin

4 .3 Analysis of caps following enzymic digestion 4.3.1 Chromatography on DBAE- cellulose and DEAE- cellulose Dihydroxyboryl aminoethyl cellulose (DBAE) is a derivative of DEAE-cellulose containing covalently-bound dihydroxyboryl groups. Its use as an affinity matrix depends upon the selective affinity of 2 1 -3 1 cis-diols for dihydroboryl groups. DBAE-cellulose was developed originally for the isolation of 3 1 -terminal oligonucleotides from high molecular weight RNA (45). However, it can also be used to isolate the 5 ' -terminal capped oligonucleotide from RNA digests since the cap possesses a 2 ' -3 1 cis-d.iol on the ribose moiety of m7 G. The procedure is described in Protocol 12. Caps removed from mRNA by enzymic digestion (Section 3.2) may also be separated by chromatography on DEAE-cellulose. Protocol 13 describes the initial separation of caps, which elute from DEAE-cellulose between 0.2 M and 0.25 M NaCI, and subsequent concentration and desalting of the caps. Protocol 12. DBAE-cellulose chromatography Equipment and reagents • Application buffer (1.0M NaCI, 0 . 1 M MgCI2, 20% DMSO, 50 mM morpholine. pH 8.71

• Capped mRNA digested with RNase T2 as described in Protocol 9 ; this will be -0.5 mg/ml in 10 mM sodium acetate, pH 4.5, 1 mM EDTA

• Application buffer plus 1.0 M sorbitol (J . T . Baker)

e o.5 M morpholine buffer, pH 8.7 • 5.0 M NaCI

e DBAE- cellulose column (15 x 0.6cm) equilibrated in application buffer

• 1.0 M MgCI 2 • Dimethylsulphoxide (DMSO; Pierce HPLC, spectrograde)

Method 1 . Adjust the composition of the RNase T2-treated starting sample to that of application buffer (buffer A) as follows :

• RNase T2-digested capped mRNA

250 ~I

• 0. 5 M morpholine, pH 8 . 7

100 ~I

• 5.0 M NaCI e 1.0M MgCI 2

200 111

• DMSO

200~1

100 ~I

• H2 0 to 1 ml final volume 2 . Apply the sample to the equilibrated DBAE- cellulose column. 3 . Develop the column with application buffer at a flow rate of - 5 ml/h and collect 1 ml fractions.

Continued

57

2: Capping and methylation of mRNA Protocol 12. Continued 4. Assay -

25~-JI

aliquots of each fraction by scintillation counting.

5 . After most of the 3'-nucleotides (resulting from the digested mRNA) have eluted from the column, change the elution abruptly to application buffer plus 1.0 M sorbitol and continue the elution at a flow rate of -6-7 ml/h. 6 . Combine the fractions containing the caps (they elute within - 1 0 ml after changing to the sorbitol-containing buffer) and proceed to the next procedure, e.g. DEAE-cellulose chromatography as described in

Protocol 13.

Protocol 13. DEAE-cellulose chromatography

Equipment and reagents • Caps isolated by DBAE-cellulose chromatography (sorbitol-eluted fractions from Protocol 12, step 61

and 1.0 x 0.6 em) equilibrated with 20 mM Tris- HCI, pH 8.0, containing 7 M urea • Yeast tRNA digested w1th pancreatic RNase; incubate 1 mg/ml tRNA• with 0. 1 mg/ml of pancreatiC RNase for 6 h at 37 ° C m 0.1 M Tris-HCI. pH 8 .0

e 1.0 M Tris-HCI, pH 8.0 e 50 mM NaCI in 20 mM Tns-HCI, pH 8.0. containing 7.0 M urea e 0 . 3 M NaCI in 20 mM Tris-HCI, pH 8.0, containing 7.0 M urea • DEAE-cellulose columns (40 x 0 .6 em

• A senes of freshly-prepared ammonium bicarbonate solutions (50 mM, 75 mM. 0 .1Mand1.0M)

Method 1. Dilute the combined fractions of the sorbitol-eluted caps 10 -fold with

H2 0. 2. Add, as a marker for the oligonucleotide elution posit ion, 0 . 5 ml (- 20 A 260 units) of a pancreatic RNase digest of yeast tRNA. 3 . Apply the mixture to the DEAE-cellulose column and wash the sample in 20 mM Tris - HCI, pH 8 .0 , containing 7 M urea. 4 . Elute the column with a linear salt gradient of NaCI (50 ml each of 50 mM and 0.3 M NaCI in 20 mM Tris-HCJ. contai ning 7 M urea). 5 . Monitor the eluate continuously at A 260 (or by scintillation counting if radiolabelled yeast tRNA has been used) for mononucleotides (- 2 charge). dinucleotides (- 3 charge ). etc. 6 . Collect the fract1ons containing caps (position of -4 to -6 charge, depending on extent of 2'-0-methylation). 7 . Desalt and concentrate the samples containing the caps by DEAE chromatography by diluting the combined fractions 10-fold with H20 and applying the pooled sample to a small DEAE column (0.6 x 1 em).

Continued 58

Yasuhiro Furuichi and Aaron ]. Shatk:in Protocol 13. Continued 8. Wash the column successively with 5 ml each of the freshly prepared 50 mM , 75 mM, and 0.1 M NH 4HC03 solutions. 9 . Finally elute the caps with 1 ml of 1 M NH 4 HC 0

3

and lyophilize the eluate.

• Radiolabelled tRNA may be used.

Figure 5 illustrates the combined use of DBAE-cellulose and DEAE-cellulose chromatography. In this example, 32P-labelled avian sarcoma virus RNA (ASV; 1.1 x 107 c. p.m., specific radioactivity 1.5-3.0 X 107 c.p.m ./~g) was isolated from purified virus (46), mixed with [3H] methyl-labelled reovirus mRNA synthesized in vitro (see Protocol I) (I 0 ~g. I x lOS c. p.m.) and digested with 50 units of RNase T2 (see Protocol 9). After incubation for 6 h at 37 °C, the mixture was applied to a DBAE-cellulose column (see Protocol 12). After the decapped 32 PIabelled ASV RNA had been removed by washing the column with application buffer, the column was eluted by applying buffer containing I M sorbitol (see Figure 5A) as described in Protocol 12, step 5. The 32P-Iabelled caps eluted in the same fractions as the [ 3H]methyl-labelled reovirus mRNA caps, immediately after the buffer change. The combined fractions containing the caps were then analysed by DEAE-cellulose chromatography (Figure 58) as described in Prorocol 13. A major peak of radioactivity with a net charge of about - 4.5 , which corresponds to the Cap 1 structure (m7GpppGmpCp), was resolved by this procedure. A minor peak eluted later with a net negative charge of about - 5.5; it corresponds to Cap 2 structures containing the ring-opened derivative of m7G (m7'G) which has lost the+ charge (46). These values are based on theoretical charges of + I at the N7 position of m7G, -3 for GpppNm, and approximately - 3 and -4 for pNp and pNmpNp, respectively. As shown in Figure 68 (inset), m7GpppGm elutes from DEAE-cellulose with a net charge between -2 and - 3, based on the elution of nucleotide markers. The divergence from the theoretical charge of - 2 ( + 1 - I - I - I) may be due to the stacking effect of the nucleosides in m7 GpppGm, resulting in stronger binding to the DEAE-cellulose matrix.

4.3.2 Chromatography on paper Cap structures and their derivatives can be resolved by descending paper chromatography on Whatmann 3MM paper in isobutyric acid: 0.5 M NH 40H 00:6 v/ v) as described in Prorocol 14. The R F values of caps and their derivatives in this system are: m7GpppAm, 0.40; m7 Gpppom, 0.36; GpppA, 0.25; GpppC, 0.16; GpppG, 0.08; and GpppU, 0.08. It is important to note that m 7G residue of the cap is sensitive to alkaline conditions and is degraded to products that include the 'ring-opened' form in which the 8-9 bond of the m7G imidazole ring is cleaved with an accompanying loss of the positive charge. Therefore, it is not appropriate to use alkaline solvents for the characterization of caps. 59

2: Copping and methylation

of mRNA

A 24

I

I

1

r8ufltr A

20-

..- 16 I

0

~

~

)(

12~

Bulftr A • Sor!Htol

~

~

RNase T2 digested ASV RNA

.........-

Caps

E ~ a.

4-

\

0

I

20

0

40

60

10-

-

8

-

6 E

0 ~

)(

.

0.

-

c. sr--

..

-

-

4 0 :::

2

eh

Fraction No.

B

..

..

I 0

I 0

~

~

)(

><

..

e

e

c.

c.

0

0

.

..:z:

..

1:1.

120

Fraction No.

Figure 5. Separation of capped oligonucleotides by column chromatography on DBAEcellulose and DEAE- cellulose. ASV genomic RNA (purified from B77-ASV that had b< m 32 P-Iabelled in chick embryo fibroblasts) was mixed with reovirus mRNAs labelled in VI •ro usmg (methyi-3 H) AdoMet (see Protocol 11. After digestion wtth RNase T2 (see Protoco. 91. the caps were separated from the 32P-Iabelled nucleotides by chromatography on DB• Ecellulose (panel A) as described in Protocol 12. The pooled cap fracttons tndicated w lre further purtfted and resolved by DEAE- cellulose column chromatography (panel Bl as describ• d in Protocol 13 into Cap 1 structures (m7 GpppG"'pCpl and the slightly more negatlv&IV chargEld Cap 1 structures which contained the ring-opened form of m 7G lm 7 'GpppG"'pCp). The approxtmate net negattve charge is indicated by the scale in panel B. Data taken fr '"" ref 4Ei. Open circles, 3 2P-Iabelled nucleotides and caps from ASV RNA; closed ctrcles. 3 [ H )methyl-labelled reowus mANA cap (m 'GpppG"'pCpl.

60

Yasuhiro Furuichi and Aaron ]. Shatkin Protocol 14. Paper chromatography of caps

Reagents • Radtolabelled caps removed from capped mANA by nuclease P1 digestion (see Protoco/9) e m 7 GpppG, m 7 GpppG"', m 7GpppA, m 7GpppA'" markers {Pharmacia-LKBI • Nucleoside and

nucleotide

(Boehringer Mannheim) as appropnate for the particular separation • Isobutyric ac1d

e 0 . 5 M NH.OH

markers

• Whatman 3MM paper and chromatography tank

Method 1. Mix the radiolabelled cap sample with authentic marker compound(s) in H 2 0. 2. Spot the mixture on Whatman 3MM paper and also separately spot samples of the desired marker(s). 3. Develop the paper by descending chromatography in isobutyric acid: 0.5 M NH 4 0H (10:6 v / v).

4. When the solvent front approaches the end of the paper (- 40 em). nemove the paper from the tank and air dry it. Locate the marker compounds under UV light and circle their positions using a pencil. 5 . Cut the paper into - 1 em horizontal strips. Count the strips to locate the radiolabelled cap.

Figure 6B illustrates paper chromatography of cap structures using the procedure described in Protocol 14.

4.3.3 Thin-layer chromatography on PEl-cellulose Perry er a/. (47) have described in detail the separation of a mixture of m7GpppAp and m 7GpppGp, prepared from RNase T2 digests of 32F'-Iabetled Trypanosoma brucei poly(A) + RNA, by PEl cellulose thin-layer chromatography using 2M pyridirnum formate, pH 3.4, as the solvent. PrOTocol 15 describes this technique. Figure 4B shows typical data obtajned by this method. Protocol 15. Analysis of caps on PEl-cellulose

Equipment and reagents • Caps derived from mANA by nuclease digestion (see Protocol 9 ) preferably 32PIabelled for ease of detection • PEl-cellulose thin-layer plates (Sigma) and TLC tank • 0 .6 M UCI or 2.0 M pyridinium formate, pH 3.4 (see step 2)

61

• Marker authentic caps. nucleostdes, and nucleotides (see Protocol 14 I • UV light source

Conlinued

2: Capping and methylation of mRNA Protocol 15. Continued Method 1. Spot a sample of the caps near one end of the plate (origin). Separately spot marker compounds along the same origin. 2. Develop the plate with 0 .6 M LiCI as solvent if the caps are denved from nuclease P 1 digestion and correspond to m 7GpppNm structures (see Figure 48). Alternatively, for RNase T2 derived cap structures (m7 GpppNp) use 2.0 M pyridinium formate , pH 3.4, as the solvent. 3. When the solvent front is near the end of the plate, remove the plate from the tank and air dry it in a hood . 4. Locate the markers by UV light and the sample spots by autoradiography or counting.

4.3.4 Paper electrophoresis

Nuclease PI digests of capped mRNAs may be analysed by electrophoresis on DES! paper or Whatman 3MM paper. The procedure is described in Protocol 16. Under the conditions described, the electrophoretic migration positions of the different caps relative to 5 ' pU are: m7GpppAm, 0.38; m 7GpppGm, 0.40; GpppA, 0.50; GpppG, 0.52; m7GpppAmpo, 0.26; and m7 GpppGpAmpC, 0.24.

Protocol 16. Paper electrophoresis of caps on DE81 paper Equipment and reagents • Caps derived from mANA by nuclease P 1 digestion (see Protocol 9) preferably 32 P-Iabelled for ease of detection •Whatman DE81 paper • Electrophoresis buffer (1% pyridine,

10% acetic acid. pH 3.5, 1 mM EDTAl • Marker compounds Including authentic caps lsee Protocol 14) • Apparatus for one-dimensional paper electrophoreSIS

Method 1. Spot a sample of the caps near one end of a 20 x 20 em sheet of the paper (origin) with marker compounds spotted in adjacent positions. 2 . Run the electrophoresis at 500 V for 2 h using electrophoresis buffer 3. Locate the compounds under UV light and by scintillation counting of 1 em slices of the dried paper.

Figures 3 and 4A show the separation, by electrophoresis on DE81 paper, of cap structures obtained by decapping and recapping a variety of RNAs.

62

Yasuhiro Furuichi and Aaron J. Shatkin A

-- - -

pCpA I 0

pU

pG

~

I 0

lOriQI"

.....

.....

m~PPI>G.,

X

X

e

e

c.

c.

u

u

..:c

c..

N

"' 0 0

0 10

20

30

B

=l

-..L....._

........ 2

X

0

2 ~

6.....

0.

-

2

ft'ITGp9pG"' ~ I

Gpj>p(l

-

:;;0..

0

6

4

E2

C.. I

:::

I l

l

o 3

-4

-3

-2

7

I 0

..... X

10

20

30

0

rracuon no

X

N

e c.

u

• I

e

:c

c.

"'

u 0 0

10

20

30

40

Fraction No. Figure 6 . Separation of cap structures by paper electrophoresis and paper chromatography. A mixture of 5'-terminal fragments derived from 32P-Iabelled ASV RNA and [ 3 H ]methyl-labelled reovirus mANA was obtained by RNase T2 digestion and DBAE-cellulose chromatography as in Protocol 12 and Figure 5A . After desalting as described in Protocol 13 (steps 7 -9). the sample was successively digested with nuclease P 1 and alkaline phosphatase and analysed by high-voltage paper electrophoresis in pyridine acetate buffer, pH 3.5, at 50 V/cm for 50 min (see Protocol 16) (panel A). The radioactive material migratmg in the position of authentic 7 m GpppG"', between the marker nucleotides 5' -adenylic acid (pA) and 5'-guanyhc acid (pG). was extracted with H 2 0 from the dried paper. and chromatographed on Whatman 3MM Paper us1ng isobutyric acid: 0.5 M NH4 0H (10:6 v/v) as described in Protocol 14 (panel B). The material in the pos1tion of the marker m 7GpppG"' was extracted with H 20 from the dried paper and an aliquot was analysed by DEAE-cellulose chromatography according to Protocol 13 (panel B. inset). Visible amounts (A 26anm -0.5) of pC. pA, pG, and pU (in panel A ) and GpppG and m 7GpppG"' (in panel Bl were included as standard markers in the separation. In addition. the ratio of 32 P: 3 H (c.p.m.) was plotted to show the homogeneity of the sample (panel B. solid circles and line).

63

2: Capping and methylation of mRNA

pA pm7d.,. /

f; pC

pGm ... )

pum ,::~····'

(::: pU

pG

Figure 7. Two-d1mens1onal thin-layer cellulose chromatography of cap constituents. Relat 1e mobilitJes are shown for m 7Gp, 2'-0-methyl-5'-nbonucleotides, and 1norgamc phosphate (P,J analysed on an Av1cei-SF cellulose thm-layer plate (431 The numbers and arrows indicate the order and d1rect1on of the two chromatography systems as descnbed 1n Protocol 17. e first dimension solvent was isobutyric ac•d:0 .5 M NH.OH (5: 3 vl v) and the second dimens• n solvent was propan-2-oi:HCI:water 170: 15:15 v/v/v).

n

4.3.5 Separation of cap constituents by two-dimensional

chromatography Cap constituents including nucleotides, m7Gp, and the penultimate 2' -0methyl nucleotides can be identified by two-dimensional chromatography on cellulose thin layers after digestion of the caps by snake venom pyrophosphat.l'e or tobacco pyrophosphatase (see Section 4.2.2) . The method, described 10 Protoco/17, can also be applied for the cap-containing oligonucleotide obtained from capped mRNA b> RNase T2 digestion (see Protoco/9) (48). Figure 7 shO\\S typical data. Protocol 17 . Two-dimensional separation of 2 '-0-methylated nucleotides using thin-layer cellulose chromatograph'; Equipment and reagents • Cap constituents prepared and labelled with 32P or 3 H as described in Protocols 7- 4, 6 - 8 and d1gested wit h nucleotide pyrophosphatases as described in Protocols 10 and 11

• Cellulose thm layer plate IAv1ceii-SF cellulose thin-layer plate. 15 x 15 em) and TLC tank

Continued

64

Yasuhiro Furuichi and Aaron ]. Shatkin Protocol 17. Continued • Isobutyric ac1d:O. 5 M ammomum hydrOXIde (5;3 V' V)

• Marker compounds !see Protocol 14)

• Propan-2·ol:conc. HCI:water (70·15 15

v'v vl

Method 1 . Spot a sample of the radiolabelled digested cap near one corner of the cellulose thin-layer plate (origin). Also apply a sample of markers to the origin. 2 . Develop the plate in the first dimension by ascending chromatography using Isobutyric acid:O. 5 M ammonium hydroxide (5:3 v/v) as the solvent.

3 . Dry the plate in a hood at room temperature. 4 . Turn the plate through 90 ° and develop it in the second dimensiOn by ascending chromatography with propan-2-ol:con. HCI water 170: 15: 1 5 v /v/v) as the solvent. 5. Locate the marker compounds under UV light and c~rcle them with a penc1l.

6. If the sample contains 32P, autorad1ograph the plate. or 7 . If the sample is [methyi-3 H) ·labelled, scrape portions of the cellulose layer off with a fine spatula and count the radioactivity using a scintillation counter.

4.3.6 Other methods Capped mRNAs and caps have also been isolated by affinity chromatography on m-aminophenylboronate agarose (49) (as an alternative to the DBAE-cellulose chromatography described in Protocol 12) and with a m7 G-specific antibody Immobilized on Sepharose (ref. 50), a matrix which is not available commercially but may be prepared as described in Volume I , Chapter 5, Protocols 1-3.

Acknowledgements The authors thank Dr Kiyohisa Mizumoto (Kitasato University, Tokyo, Japan) for valuable discussion and Akiko Fujii and Janet Hansen for excellent secretarial assistance.

References I. 2. 3. 4. 5.

Shatkin, A. J . (1976). Cell, 9, 645. Banerjee, A. K. {I 980). Micro bioi. Rev.. 44, 175. Maniatis, T. and Reed, R. (1987). Nature, 325, 673. HsuChen, C.-C. and Dubin, D. T (1976) Nature, 264, 190. Mattaj , I. (1986). Cell. 46, 905.

65

2: Capping and methylation

of mRNA

6. Agabian, N. (1990). Cell, 61, 1157. 7 Bangs, J . D., Crain, P. F., Hashizume, T, McCio~ke}', J. A., and Boothroyd, 1. C. ( 1992). J. Bioi. Chern., 267, 9805. 8 Furuichi, Y., Lafiandra, A., and Shatkm, A. J. (1977). !Vature. 266, 235. 9. Green, \I R , \1aniatis, T ., and Sharp, P i\. (1984). Cell, 38. 731. 10 Konarska, ~1. M., Padgett, R. A., and Sharp, P A. (1984) Cell, 38. -31. II. Ohno, M., Sakamoto, H ., and Shimura, Y (1981). Proc '\at/ Acad. Sci. US. 84, 5187. 12. Shatkin, A. J. (1985). Cell, 40, 223. 13. Hagler, J. and Shuman, S. (1992). Scrence, 255, 983. 14 Shuman. S. (1985). J. Bioi. Chern., 26-', %90. 15. Cong, P . and Shuman, S. (1992). J. Bra/. Chem., 267, 16424. 16. Higman, f\.1. A., Bourgeois, N.. and Nile;, E. G. (1992). J Bwl. Chem., 267, 16430. 17. Shuman, S. and "foss. B. (1989). J Bioi. Chem, 264, 21 356. 18. Beaton, A. R. and Krug, R. M. (1986) Proc Nat/ Acad. Sci. USA, 83, 6282 19. Georgiev, 0., Moss, J., and Birnstiel, \1. l (1984). Vue/. Acids Res., 12, 85 9. 20. Schnierle, B.S., Gershon, P. D.. and Moss. B (1992). Proc '\'at/ Acad. Scr. USA, 89, 2897. 21. Miura, K.-1., Watanabe, K., Sugiura, M., and Shatkin, A. J . (1974). Proc. 1\arl Acad. Sci. USA, 72, 3979. 22. Furuichi, Y, Morgan, M., Muthukrishnan, S., and Shatkin, A. J. (1975). Proc. Nat/ Acad. Sci. USA. 72, 362. 23. Furuichi, Y. and Miura, K.-1. (1975). Nature, 253, 374. 24. Furuichi, Y. and Miura, K. (1973). Virology, 55, 418. 25. Shatkin, A. J . and Lafiandra, A. J. (1972). J. Virol., 10, 698. 26. Joklik, W. K. (1962). Biochim. Biophys. Acta, 62, 290. 27. Furuichi. Y. and Shatkin, A. J. (1976). Proc. Nat/ Acad. Sci. USA, 73, 344tl 28. Furuichi, Y. and Shatkin, A. J. (1977). Nucl. Acids Res., 4, 3341. 29. Wei, C. M. and Moss, B. (1975). Proc. Nat/ Acad. Sci. USA, 72, 318. 30. Aviv, H . and Leder, P. (1972). Proc. Nat/ Acad. Sc1. USA, 69, 1408. 31. Kreig, P . A . and Melton, D. A. (1987). In Methods in enzymology, Vol 55 (ed. R. Wu). p.397, Academic Press, San Diego 32. Mizumoto, K., Kaziro, Y., and Lipmann, F (1982). Proc. Nat/ Acad. Scr. USA. 79, 1693. 33. ltoh, N., Mizummo, K., and Kaz.iro, Y. (1984). J. Bioi. Chern., 259, 1392 34 Wang, D. and Shatkin, A. J. (1984). "'11cl. Acids Res., 12, 2303 35 Yagi, Y., Mrzumoto, K., and Kaz.iro. Y (1984). J. Bioi. Chem., 259. 4695. 36. Barbos. E. and Moss, B. ( 1978). J. Bioi. Chem., 253. 7698. 37 Handa, H., M1zumoto, K., Oda, K.. Okamoto. T. and Fukusawa, T. (1985). Gene. 33, 159. 38. Shmslu, H ., Miwa, M., Sug.irnura, T .. Shimotohno, K.. and Miura, K. (1976). 1-~BS Leu.. 65, 254. 39 Zan-Kowalcze\\Ska, ~1., Bretner, \1., Sieralowska, H., Szczesna. E., Filipowicz.\\'.• and Shatkin, A. 1. (1977). 'Vue/. Acrds Res., 4, 3065 40. Stevens. A (1988) . .\folec. Cell. Bioi., 8, 2005. 41 Nuss, D. L., ruruichi, Y., Koch, G., and Sha1km, A. 1. (1975). Cell, 6. 21. 42 'luss. D. L. and Furuichi, Y. (1977). J. Btol. Chem .• 252, 2815

66

Yasuhiro Furuichi and Aaron ]. Shatkin

.n.

44. 45. 46. 47. 48.

49. 50.

i~hjmura , S. (1979). In Transfer R1 A: structure, properties, and recognition (ed. P. R. Schimmel, D. Soli, and J. '1. Abelson), Appendt>.. Ill, Chromatographic mobilities of modified nucleotides, p. 551. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Ef,tratiadis, A., Vournakis, J. N., Donis-Keller, H ., Chaconas, G., Dougall, D. K., and Kafatos, F. C. (1977). Nucl. Acids Res., 4, 4165. Rosenberg, M. (1974) .•Vue/. Acids Res .. 1, 653. Furuichi, Y., Shatkin, A. J., Stavnezer. E., and Bbhop, J. M. (1975). Nature, 257, 618. Perr}, K. K., Watkins, K. P., and Agabian, N (1987). Proc. Nat/ Acad. Sci. USA, 84, 8190. Furuichi, Y., Morgan, :\1 ., Shatkin, A J., Jehnek, \\., Salditt-Georgieff, M., and Darnell, J. E. (1975). Proc. Nat/ Acad. Sci. USA, 12, 1904. Wilk, H.-E., Kecskemethy, t ., and Schafer, K. P. (1982}. Nucl. Acids. Res., 10, 7621. Munns, T. W., Oberst, R. J., Sims, H. F., and Liszewski, M. K. (1979). J. Bioi. Chern., 254, 4327.

67

RNA editing in mitochondria LARRY SIMPSON, AGDA M. SIMPSON.

and BEAT BLUM

1. Introduction 1.1l Post-transcriptional modification of eukaryotic mRNAs Several examples of specific modifications of nucleotide sequences within coding regions of mRNAs, and, in some cases, within rRNAs, have been observed in recent years. This process, which is known as RNA editing, ranges from the single C- U substitution in the mammalian apolipoprotein B (1, 2), to the multiple C-+ U substitutions found at specific sites in plant mitochondrial mR.NAs (3, 4), the multiple C insenions found in mitochondrial mRNAs andl rRNAs in Physarum polycephalum (5), and the multiple U insenions and dele!tions found in the mitochondria of kinetoplastid protozoa (6). The use of the term, RNA editing, for these various types of modifications does not necessarily imply that the mechanisms are identical. In fact, the polymerase 'stUittering-induced' addition of extra G residues at specific sites in mRNAs from neg,ative strand RNA viruses is also termed 'RNA editing' (7). The editing occurring in the mammalian apolipoprotein B and mRNA appears to be due to a site-specific enzymatic deamination of cytidine (8-13), but nothing is known about the mechanism of the multiple C-+ U transitions in plant mitochondria. Likewise, nothing is known about the mechanism of C-insertion in Physarum mitochondrial RNAs. The best understood case is kinetoplastid RNA editing which is described below.

1.2 RNA editing in kinetoplastid protozoa An unusual type of RNA processing occurs in the single mitochondrion of kinetoplastid protozoa. Many of the mRNA transcripts of structural genes enC'oded in the maxicircle DNA molecules are modified within coding regions in a post-transcriptional process characterized by the insertion and, less frequently, the deletion of uridylate (U) residues (14). This process, known as RJI.IA editing, involves small RNA molecules, guide RNAs (gRNAs), which specify the sequence information required. Two models have been proposed for the mechanism of this process (15, 16). Both models propose an initial

3: RNA editing in mitochondria base-pairing interaction bet ween the 5 ' -end of a spec1fic gRNA and the mRNA immediately 3 ' to the pre-edited region (PER) to form the 'anchor' hybrid The enzyme cascade model then proposes a specific cleavage at the first mismatched nucleotide of the mRNA, followed by the addition of a U residue to the liberated 3 '-end, and an RNA ligation to seal the mRNA backbone. Eacr U residue added extends the initial hybrid between the mRNA and gRNA b forming an additional base pair to either A or G 'guiding' nucleotides o the gRNA. This process is repeated until a complete hybrid between gRNA and mRNA is ach1eved. The trans-esterification model (16, 17) proposes that the cleavage-ligation occurs by means of two successive trans-esterification steps The fir~t of these imolves the 3' -terminal oligo(U) tail of the gRNA as the sourct of the Us for transfer. Formation of gRNA:mRNA chimaeric molecules attached within the PER has been observed both in l'ii'O among kinetoplast RN -\ (kRNA) molecules (16) and in vitro using mitochondrial extracts (18, 19 Formation in vitro is dependent on complementary anchor sequences in the gRNA and mRNA (20). However, the available evidence does not allow .. distinction to be made between the chimaeric molecules formed by rran esterification or by cleavage-ligation. The trans-esterification model is artractr e due to its close analogy with RNA splicing. RNA editing is restricted to genes encoded by the mitochondrial genome whiLh consists of a network of catenated DNA minicircles and maxicircles. T r ~ network, referred to as kinc!toplast DNA (kDNA), is contained within the sint;le mitochondrion which reticulates throughout the entire cell and is located in close proximity to the basal body of the flagellum (21 ). The genes for the mitochondrial rRNAs and a set of mitochondrial structural genes, several of which undergo RNA e·diting, are encoded on the maxicircle DNA. The maxicirclc DNA of Leishmania tarentolae consists of 20-50 copies of a 30 kbp circular molecule. Howevt:r, the bulk of kDNA comprises minicircles, which are present in high copy number (approximately I 0 4 molecules per network) and display multiple sequence classes. In L. rarentolae each copy of the D~A minicircle is organized into a conserved region of approximately 170 bp and a variable region of approximately 700 bp which defines the sequence class. Ea~h sequence class encodes a single unique gRNA located approximately 150 bp from the end of the conserved region (22). In other kinetoplastid species, the number of conserved regions 'aries from one to four. In Trypanosoma brucet there is a single conserved region, but the variable region encodes three gRNA gene) (23). A region of 'bent' DNA of unkno,\n function exists either adjacent to the conserved region in the minicircles of L. rarenrolae and T. brucei. or at 90 ° to the conserved region in the larger minicircles of Cmhidia fascicula t1 The presence of this bend decreases the electrophoretic mobility of the D~ .\ in polyacrylamide gels as compared to agarose gels and is partially respom"ble for the increased complexity of the polyacrylamide gel profiles of restriction enzyme-digested kDNA (24). The complexity of minicircle DNA varies between kinetoplastid species. Some species, which lack the ability to li\'e in an insect

70

Lorry Simpson, Agda M. Simpson, and Beat Blum host, ha\e minicircles of a single sequence class. The kDNA of T. brucei contains approximate!} 300 different minicircle sequence classes; the kDNA of L. tarencolae comains at least 17 different sequence classes in differing abundances (25). The complexity of restriction enzyme-digested kDNA, as visualized by electrophoresis in gradient polyacrylamide gels, can be used to classify and type different strains within a species, at least for Leishmania and Trypanosoma cruz1. The term, sch1zodeme. was coined to indicate organisms with similar kDNA restriction proftles (26, 27). The kDNA restriction profile is a very useful molecular marker since strains are frequently mislabelled, even some a\ailable through the American Type Culture Collection (28). In this chapter, procedures are described for the grO\\ th of kinetoplastid cells, isolation of the kinetoplast-mitochondrion fraction, isolation of kDNA and kRNA, identification of gRNAs by computer analysis, isolation of gRNAs by hybrid selection, and assays for se\eraJ mitochondrial enzymes possibly involved in RNA editing including the terminal uridylyl transferase (TUTase), an RNA ligase, a cryptic RNase, and a gRNA:mRNA chimaeric-forming activity.

2. Growth and maintenance of kinetoplastid protozoa

2.1 Choice of species for experimental work The kinetoplastid protozoa comprise a large group of parasitic flagellated cells with a single multilobular mitochondrion containing the kDNA network of catenated circular DNA molecules (29). The kinetoplastid flagellates belong to 8- 10 genera, several of which are digenetic with a life cycle that involves successive vertebrate (or plant) and invertebrate hosts, and several of which are monogenetic with a life cycle in a single invertebrate host. The monogenetic species such as Critludia or Leptomonas have simpler nutritional requirements than the digenetic species such as Trypanosoma or Leishmania. In the latter case, the stage of the cycle in the insect vector is generally easier to culture than the stage in the vertebrate host. Phyromonas is a digenetic species which inhabits a plant host and an insect vector. Unlike the other digenetic species, the stage from the plant host grows readily in simple media and may provide a model system for the study of metabolic changes occurring during the life cycle (F. Opperdoes, personal communication). RNA editing has been studied in three species to date: T. brucei, L. tarentolae, and C. fasciculara (6, 14, 29-31 ). The major differences between these protozoa involve the complexity of the minicircle DNA population and the correlated extent of ediling of the three cryptogenes-ND7, CO III, and MURF4. In addition, regulation of RNA editing has been observed to occur during the life cycle of T. bruce1 (32, 33). The authors have chosen to use the saurian leishmania, L. tarenrolae, as a model system to study RNA editing for the following reasons: 71

3: RNA editing in mitochondria (a) The cells are not pathogenic for humans, which is a major advantage in terms of growing large amounts of cells in the laboratory for the isolation of mitochondria, nucleic acids, and enzymes. (b) The cells grO\\ rapidly (6-9 h division time) in brain heart infusion (BHT) medium, an easily prepared rich medium, without the need for serum supplementation.

(c) The cells can be frozen for storage. (d) Cell concentrations of 4 x I OS ceils/ ml can be obtained at stationary phase {e) The cells can be ruptured, after swelling in hypotonic medium, to yield an intact ki netoplast-mitochondrion fraction which is active in transcription and possibly also in RNA editing. (f) Several recently-isolated strains exist for comparative studies.

(g) The minicircle DNA and, therefore, the gRNA complexity is limited, makint, it feasible to determine a complete list of gRNAs for all the genes know to be the subjects of RNA editing. In contrast, the other two protozoa in "'hich RNA ediung is known to occL. have some disad.,antages: (a) The procychc cells of T. bmcei require complex media with serum and c1o not reach high cell densities. The bloodstream cells must be grown eithtr in a rodent host or in a complex medium in which they only reach low cell densities (34). (b) The main disadvantage of C. fasciculata is that the cells swell poorly in hypotonic buffers and do not break by the use of shear forces alone. On the other hand, they grow extremely easily and to high densities in BH I medium and also in a defined medium. They also plate on agar surfaces more easily than either L tarentolae or T. brucei. The major dtsadvantage of working with L tarentolae is that the biology of the parasite within the natural lizard host is essentiaJiy unknown. Theref01e, one cannot stud} the vertebrate stage of the life cycle. It is clear. howe\'er, 1at the leishmania which infect lizards form a subgroup of the genus Leishmania (28, 35), which also contains the mammalian pathogenic leishmania such ;u L major, L. mexicana, and L. brasiliensis.

2.2 Growth and maintenance of L. tarentolae Initial stocks of L. tarentolae (UC strain) can be obtained from Dr L. Simpson. Howard Hughes Medical Institute, UCLA, Los Angeles, CA 90024. Protocol / describes the maintenance and storage of stock cultures. To avoid accumulauon of genotypic changes by prolonged culture, begin a new stock culture from frozen stocks every few months.

72

Larry Simpson, Agda M. Simpson. and Beat Blum Protocol 1 . Growth and storage of L. tarentolae stock cultures

Equipment and reagents • 2 mg ml haem1n: add 2 mg haemtn tSigma H-23751 per ml of 50 mM NaOH sttr for 30 m1n, and stenhze by filtrat•on through a 0.22~Jm membrane l-Iter INalge); store at - 20 °C

htre of t1ssue culture grade water auto clave in Pyrex bottles at 1 20 °C for 30-40 min' when cool. aseptically add haemin to 10 mg1ml; the med1um ts stable for several months at 5 °C

• Tissue culture grade water (prepared using Barnstead Nano..pure or Millipore Milli·Q cartridge filtration system) (Barnstead Co ., Millipore Corp.)

• BHI medium contatning 20% glycerol, sterilized by autoclaving tn Pyrex bottles

• Pyrex bottles 500ml IF1sher. #06 414- lC) with screw caps (Ftsher, d 06-414-2Al. for autoclaving solut•ons • Brain heart tnfusion IBHII medtum•: dissolve 37 g BHI powder tD•fco) per

• Tissue culture flasks (glass; Corning #25100; 25cm2 ) • Inverted phase-contrast microscope • Freezer v1als. stenle, polypropylene }. ml IVan Waters and Rogers ~ 66008·3091

Method A. Mamtenance and momtormg of cultures 1. Inoculate healthy !mottle) L rarenrolae cells at 0.6-1.5 5-10 ml BHI medium m 25 cm 2 tissue culture flasks.

106 cells fml in

2. Grow the cultures at 27 °C 3 . Check the growth of the cultures daily using an inverted, phase -contrast mtcroscope . Cultures can be monitored more accurately for the absence of bacteria by screening wet-mount slides under phase-contrast microscopy at 400 x or 1000 x magnification. Leishmania cultures have a characteristic wave-like appearance to the eye that ts qUite different from the homogeneous turbtd appearance of bacterial cultures. Also the smell of the healthy cultiJre ts frutty and qutte charactensttc 4 . Every 2-4 days, aseptically remove most of the culture leaving about 0 05-0. 1 ml and add 5 ml of fresh BHl medium.

B. Storage and recovery of cells 1. Dtspense 0 .5 ml aliquots of healthy log-phase cultures (approxtmately 108 cells mil into sterile 2 ml freezer vials. 2 . Add 0 5 ml of BHI medium containing 20% glycerol 3 . Place the vtals 1n a 1 litre beaker filled with cotton wool and put the beaker m a - 70 °C freezer. This allows slow freezing of the cells. 4. The next day transfer the vials to liquid nitrogen for long-term storage. 5 . Recover live cells from stored cultures by thawing the vials rapidly and inoculating the contents into 2-5 ml of BHI medium. Check the cells by phase-contrast microscopy; they should be motile tmmediately after thawing. • No antibiotics are necessary if aseptic techniques are used and stenlity 1s preserved , • The medium can be autodaved dtrectly 1n glass culture bottles or in glass bottles for storage

73

3· R\'A editing in mitochondria

2.3 Cloning of kinetoplastid protozoan stock cultures !\lost laborator) kmetoplastid stocks are uncloncd and may contain several different strains. Soon after receipt, cells should always be cloned either b} limiting dilution in microtitre plates or by growth on 0.7- I.Oo/o agar/ BHIbaemin plates and selecting single colonies. One should verify the identity of the strain or even the species by a combination of light microscope morphology and molecular characteristics. Schizodeme analysis by comparison of kDNA sequences (see Proroco/4) IS an easy method to u~e for verifying the identit> of a strain of T. cru~t or Leishmania.

2.4 Growth of cultures for preparation of kinetoplast

components Cultures up to I litre in volume can be grown in 3.81itre roller bottles using a standard roller-bottle culture apparatus at 16 r .p.m. Harvest the cells b} centrifugation at 2500 g for I 0 min at 5 °C. Grow larger cultures in a stirrer culture apparatus with fo rced aeration and harvest the cells by filtration a~ described in Proroco/2. Cells can also be grown in a microbiological fermentor with aeration and stirring. However, the addition of silicon anti foam (Anti foam B, Sigma) is required to prevent foaming. Protocol 2. Growth and harvesting of large-scale cultures Equipment and reagents • Stirrer culture apparatus (Bellco 15 litre or 36 litre bottles, # 1964·1 5000 or 36000, With overhead stirrer # 7664 -001 1 0 and stainless steel impeller If 1964-600151 • Air pump or source of compressed air fitted With a 0.2 11m Gilson microbiological !titer on input and eXit lines

• Compressed 0 2 (medical grade); required for larger cultures • Transverse filter system !Millipore Pelficon with 0.45 11m Durapore filter cassette) with 8 htres min peristaltiC pump (Millt· pore Masterflex XXBO ELO 01 I • Log-phase culture of L. rarenrolae and BHI medium as in Protocol 1

Method 1 . Inoculate BHI medium with L. tarentolae at approximately 1.2 x 106 cells/ml using a log-phase culture. For 151itre bottles use 1 80 ml of culture at a cell density of 10 8 cells/mi. 2. Stir the culture at maximum rate at 27 °C. Blow filtered air into the bottle (but not through the med1um) at 6-8 litres. min. Filter the exhaust air into a hood exhaust. 3 . If larger cell yields are desired, substitute 0 2 for a1r aher approximately 50 h. This Will allow cells to grow in log-phase up to a cell density of approximately 4 x 108 cells/mi. wh1ch is tw1ce the density reached w ith air alone.

Continued

74

Larry Simpson, Agdo .\J. Simpson, and Beat Blum Prot o col 2. Continued 4 . Harvest the cells when they have reached a density appropriate for the subsequent preparation (see later Protocols). To do th1s, cool the culture by immersing the culture bottle in ice water while stirring the culture. Concentrate the culture to approximately 100-200 ml by filtration usmg the Millipore Pellicon transverse filter system. 5 . Wash the cells by addition of the desired wash medium to the concentrated cell suspension and reconcentrate the cells. 6 . Flush the washed cells out of the filter system by a fmal rinse with wash medium. 7 . Concentrate the cells again and then pellet them by centrifugation at 2500g for 10min at 5 °C. 8 . For kDNA isolation. resuspend the cells 1n SET medium and freeze them at - 70 °C (see Protocol3). For isolation of the kinetoplast-mitochondnon fraction , process the cells immediately as described in Protocol 6.

3. Kinetoplast DNA 3.1 Introduction Kinetoplast DNA IS present in the form of a single giant network per cell composed of approximately 104 catenated minicircles and 20-50 maxicircles (21). The kDNA network has a sedimentation coefficient of 4000 S units and is relatively resistant to shear forces due to its compactness (36, 37). It is easily isolated from a sheared total cell lysate by sedimentation through CsCI (27). The maxicircle DNA represents 507o of the kDNA and can be isolated on the basis of its relatively higher A+ T content (84 117o A-'- T versus 55117o A+ T for rrunicircle DNA) after its release from tbe network by digestion with a restriction enzyme that cuts onl~ once or mfrequemly (38). The complete sequence of the 23 kbp maxicircle of T. brucei is known, and 21 kbp of the 30 kbp maxicircle genome of L. taremolae has been sequenced (Genbank entr} LEIKPMAX). The structural genes are clustered in - 17 kbp and the remainder. which is known as the ctivergent region, represents tandem repeats of differing complexities. Minicircles and maxicircles replicate once per cell cycle in an S phase which is synchronous with the nuclear DNA S phase. Minicircles are randomly decatenated, replicate the network and are recatenated at two sites at the ends of the kDNA nucleoid body (39). Replicated minicircles are 'nicked' or 'gapped', and are simultaneously covalent!~ closed early in the G 2 phase of the cell cycle. :\laxicircle D!'IA replicates by the rolling circle mechanism and a low concentration of linearized ma'\icircle intermediate molecules can be found free of the network (40). Therefore, to maximize y1elds and to obtain CO\alently closed circular DNA molecules, stationary phase cells must be used for kD A isolation. 75

3: RNA editing in mitochondria

The complexity of the minicircle DNA population is characteristic of each kinetoplastid species. Furthermore, in Leishmania and T. cruzi, a specific sequence heterogeneity within the minicircle population defines each particular strain or schizodeme. Tibayrenc eta/. (41) have suggested that the genetic reaso1 for this heterogeneity is the long-term genetic isolation of each schizodeme or clonal line without gene exchange. In the case of T. cruzi, they ha\ coined the term 'clonet' (Tibayrenc and Ayala, personal communication) to describe these lines of parasites which have been clonally derived a nd isolated for perhaps millions of years. In any case, the term 'schizodeme' an appropriate operational term to describe T. cruzi or Leishmania strains that differ in kDNA minicircle sequences.

3.2 Isolation of kDNA Protocol 3 describes the isolation of kDNA from stationary phase cultures "f L . rarenrolae. Protocol 3 . Isolation of kDNA

Equipment and reagents e SET buffer 10.15 M NaCI. 0. 1 M EDTA,

e 1010 .1 TE buffer (10 mM Tris- HCI pH 7.9, 0. 1 mM EDTA)

10 mM Tris- HCI, pH 7. 5) • Stationary phase culture of L. tarentolae; about 4 x 108 cells/ml for large aerated cultures (see Protocol 21 or 1 5-2.0 x 108 cells/ml for roller-bottle cultures • 10 mg/ml pronase (Calb1ochem); d1ssolve the pronase in SET and predigest the solution for 30 m1n at 37 to inactivate nucleases

• 1011 TE buffer (10 mM Tns-HCI, pH 7.9 1 mM EDTA) • 250 ml polycarbonate screw·capped flask • 1 0 mgtml ethidium bromide

•c

• 30% Sarkosyl: stir 30 g of sodium sarcosinate 1n 60 ml of water at 60 •c, add water to 100 ml; store the solution in aliquots at - 20

•c

• Hypodermic synnge 112 ml) fitted with an 18-gauge needle or • Dispensing pressure vessel IMillipore XX67 OOP 05 or 10, for the 5 litre or 10 litre sizes, respectively); this has an 18-gauge needle fitted v1a a luer·lock adapter to one outlet and a pressure release valve attached to another outlet; use a quick release tube adaptor to attach the vessel to a compressed air supply • Beckman ultracentrifuge SW28 rotor, and 36 ml polyallomer tubes (or eqUIValent) • CsCI (density gradient or optical grade)

• Upper CsCI solution: 37 .62 g CsCI ir 62 ml of 10/ 1 TE buffer (refractive inde~ at 25 •c = 1.3705) • Lower CsCI solution · 29 2 g CsCI II" 20 ml of 10/ 1 buffer plus 0 . 14 ml of 10 mg ml eth1dium brom1de (refractive ~ndex at 25 •c = 1.4040) ; note only th lower CsCI solution contains dye • Peristaltic pump • UV microscope

• uv lamp 1254 nm) • Butan-1 ol saturated with water • 1 2 ml polystyrene centnfuge tube • Sec-butanol • Phenol:chloroform ( 1· 1, v /v) • DAPI (4' 6 '-oiamid~no-2 phenyhndole Sigma II D 13881 e 2.0 M NaCI

Continued 76

Larry Simpson. Agda M. Simpson and Beat Blum Protocol 3. Continued

Method 1. Harvest the cells from roller cultures by centrifugation 11 0 min at 2000-3000 gat 5 °C) or by filtration for large cultures (see Protoco/2) . 2 . Wash the cells by resuspending them in at least 50 vol. of SET buffer and repeating step 1. 3 . Resuspend the cells at 1. 2 x 109 cells/ml by stirring and pipet:ting in SET buffer ensuring there are no clumps. Add 0 .02 vol. of 1 0 mg/rnl pronase and 0. 1 vol. of 30% sarkosyl. Incubate at 60 ° C for 1-3 h with occasional shaking until the lysate clarifies. 4 . Pass the viscous lysate through an 18-gauge syringe needle at 25 p.s.i.

to shear nuclear DNA selectively and decrease the viscosity. For small volumes ( 10- 100 ml) use a 12 ml syringe fitted with an 18-gauge needle and force the lysate through it by hand. For large• volumes, use a dispensing pressure vessel with the pressure supplied by compressed air. 5 . Centrifuge the lysate for 1.5 h in an SW28 Beckman rotor at 22 000 r.p .m. at 5 °C . This pellets the crude network DNA. 6. Add 1-2 ml of 10/0 . 1 TE buffer to each gelatinous pellet. Hesuspend each pellet by shaking and pour the pellets from all the tubes into a 250 ml polycarbonate screw-cap flask. Use 6 ml of 10/0.1 TE buffer per 1-21itre of original culture. Shake the pellets vigorously for 30 min to dissolve the crude network DNA. The crude network DI\IA can be stored at 5 °C. 7 . Prepare CsCI step gradients in SW28 polyallomer tubes. Slowly introduce 6 ml of the lower CsCI solution below 24 ml of the upper CsCI solution using a peristaltic pump to give a sharp interface.

8. Layer 6 ml of the resuspended crude kDNA solution (from step 6) on each gradient and centrifuge the gradients for 15 mfn at 20 000 r.p.rn. at 20 °C in an SW28 rotor. Allow the rotor to slow down with the brake on. The kDNA networks will sediment to the interface between the lower and upper CsCI solutions while nuclear DNA, RNA, and protein will remain in the upper CsCI solution. If the shearing of the lysate was insufficient, some nuclear DNA will also sediment close to the interfacEl. 9 . Visualize the kDNA band at the interface with the UV lamp. Seal the top of the tube with a rubber stopper fitted with a needle attached to a 20 ml syringe. Puncture the bottom of the tube and use the syringe to collect the kDNA band into a 1 2 ml polystyrene centrifuge tube. 10. Remove the ethidium bromide by extracting twice with an equal vol. of water-saturated butan-1 -ol. 11 . Dialyse the kDNA against 41itres of 10/0.1 TE buffer. 12. Concentrate the kDNA solution to approximately 400 Ill by several extractions with sec-butanol. Use an equal val. of sec-butanol for each extraction and the volume will be halved each time. Centnfuge for 2 min in a clinical centrifuge to break the emulsion. Transfer the 400 Ill kDNA solution to a microfuge tube.

Continued 77

3:

R~A

editing in mitochondria

Protocol 3. Continued 13. Extract the kDNA by vortexing with an equal val of phenol:chloroform (1 1) Separate the phases by centrifugation at 10 000 g for 1 min. Excess centnfugation will result

1n

loss of network DNA at the interface.

14. Remove the aqueous (upper) phase and remove traces of phenol from it by vortexing 1t 2-4 times with an equal val. of water-saturated ether. Allow the phases to separate by standing and then aspirate off the ether.

15 Add 0.01 val. of 2 M NaCI and 2 val. of ethanol. Incubate in dry icealcohol bath for 15 min or at - 20 °C overn1ght. 16. Recover the precipitated kDNA by centrifugation at 12 000 g for 14 min at 5 °C in a microcentrifuge. 17. Resuspend the kDNA pellet at 1 mg/ml in 10.1 1 TE buffer. 18. Check the integnty of the network DNA by diluting a sample ten-fold in 10/1 TE buffer and adding DAPI to 1 ~g/ml final concentration. Observe the stained kD NA using an UV microscope at 1000 x magnification. The size and shape of the liberated kDNA networks are distinctive for each species of kinetoplastid. If the shearing (step 4) was too harsh, the networks will be fragmented. L. tarentolae networks often break into half- or quartersized networks. C. fasctcu/ata networks are more stable to shear forces.

19. Store the kDNA in aliquots at -20 °C.

Using the procedure described in Protocol 3, the yield of kDNA is 0.5-1.0 mg/ litre of culture. Figure I shows the kDNA isolated from L. taremo/1 e as in Protocol 3. If desired, nuclear DNA can be isolated from the crude DNA preparation (step 6). Add 2 vol. of cold (- 20 °C} ethanol and spool the nuclear DNA onto a glass rod. Dissolve the DNA in 10 ml of 10/ 1 TE buffer. Deproteinize it by phenol extraction and recover the DNA by ethanolprecipitation as described in Protocol 3, steps 13-16.

3.3 Schizodeme-typing of kinetoplastid protozoan strains Schizodeme-typing is used to verify the strain of protozoan used. It relies on the use of speci fie restriction enzymes to cut the kDNA minicircles and maxicircle • For L. tarentolae, Haelll, Mspl, Taql, and Rsal are suitable enzymes wh1ch liberate most of the minicircles and have multiple sites in some of the mimcircle DNA sequence classes. The procedure is described in Protocol 4. Protocol 4 . Sch1zodeme-typing of strains

Equipment and reagents • kDNA prepared as descrobed •n Protocol 3 • Appropnate restriction enzymes and buffers (see supplier's instruct•onsl • Polyacrylamtde gel I 1 mm th1ck. 4. 5- 10% ltnear gradient of acrylamrde, 4 % stack)

•n TBE buffer (see Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 111; use a standard grad•ent former and pump and add 1 5% glycerol to the 10% acrylam1de solutron when formmg the gradient

Contmued 78

Larry Simpson, Agda M. Simpson and Beat Blum Protocol 4. Continued e 10 mg•ml ethid1um brom1de • 50% methanol, 10% trichloroacetiC acid • 5% acetic acid, 10% methanol • Concentrated NH4 0H

• Sliver nitrate soluuon (1 g 1n 10 ml water) • 0 .01% citric ac1d conta1nmg 0.45 ml of formaldehyde per 300 ml; make this solution fresh • 20% ethanol, 5% acetic ac1d

Method 1. Digest 41Jg aliquots of the kDNA w1th several different reS1rict1on enzymes 2. Separate the restncted kDNA on the 4. 5-10% polyacrylamide gradient gel using TBE buffer in the electrode reservoirs as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4 , Protocol 11. 3 . Visualize the DNA bands by staining the gel with either ethidium bromide (0. 51Jg/ml final concentration) or silver . For eth1dium bromide staming see ref. 42. For silver staining, see steps below•. 4 . Fix the gel in 200 ml of 50% methanol, 10% trichloroacetic acid for 30min. 5 . Wash the gel twice in 250 ml 5% acetic acid, 10% ethanol for 20 min each time, then wash twice in 250 ml 10% ethanol for 10 min each. 6 . Mix 40 ml of 0.1 M NaOH. 3 ml of concentrated NH 4 0H and 10 ml of silver nitrate solution. St1r the mixture until the black precipitate dissolves. Add water to 200 mi.

1 . Stain the gel in the silver solution (step 6) for 40 min 8 . Wash the gel with 300 ml of water for 5 min. 9 . Develop the gel with 300 ml of freshly made 0 .0 1 o/o citric acid containing 0.45 ml formaldehyde . Stop the development in 20% ethanol, 5% acetic acid for 10 min. 10. Wash the gel three times in 250 ml of 20% ethanol for 30 mm each t ime. Based on refs 43 44.

The complex restriction pattern obtained, which is mainly due to the dige~ted minicircle DNA, is characteristic of a particular sch1zodeme. Maxicircle DNA can be seen as minor high molecular weight bands. Patterns of several T. cruzi schizodemes are shown in refs 26, 27, and 45-47. Figure 2 shows the schizodeme analysis of T. cruzi strains.

3.4 Isolation of maxicircle DNA In aU kinetoplastid species analysed, maxicircle DNA, which ranges in size from 23 to 36 kbp, has a relatively high A+ T content as compared to minicircle DNA. This property can be exploited to allow the separation of maxicircle DNA on Hoechst 33258-CsCl density gradients (38). The Hoechst dye binds preferentially to (A + T)-rich sequences, thereby decreasing the buoyant density . The technique is described in Protocol 5. The linearized maxicircle DNA prepared by this method from L. tarentolae is 30 kbp in size. 79

3: RNA editing in mitochondria

Figure 1 K1netoplast network DNA from L. tarentolae. Isolated kDNA statned w1th DAPI and visualized at 1000 x magmf1cation with UV illummat1on (see Protocol 3) These cup lille structures represent monomolecular sheets of catenated montc~rcles and maxicircles.

Protocol 5. Isolation of maxicircle DNA

Equipment and reagents • kDNA, 10, 1 TE buffer, secbutanol , 2.0 M NaCI, and UV lamp (254 nml as described in Protocol 3 • Appropriate restnctton enzymes and buffers (as described in supplier s in· structionsl; use an enzyme that cuts max1circle DNA once or infrequently. EcoR 1 is su1table for L tarentolae kDNA • 0. 7% agarose geltn TBE buffer Nolume I,

Chapter 1 Protocol 6 or Volume I Chapter 4 , Protocol 121 • CsCI !optical grade) • 0 5 mg ml Hoechst dye L33258 !Sigma, B 28831 • Beckman ultracentrifuge. 50 and 50. 2 ot Ti60 rotors and tubes (or equivalent) • Pro pan 2-ol

Method 1. Dilute 1 mg of kDNA into 2 ml of EcoR 1 restnctton enzyme buffer Add 1000 units of EcoR 1 and 1ncubate the mixture for 3 h at 37 °C.

Continued

80

Larry Simpson. Agda M. Simpson. and Beol Blum

Protocol 5. Continued 2. Monitor the extent of release of the 30 kbp maxicircle DNA by runmng 101-11 samples on a 0 . 7 % agarose gel (Volume I, Chapter 1, Protocol 6 or Volume I, Chapter 4, Protocol 12) 3. Mix the 2 ml digested kDNA w1th 1011 TE buffer to 12 ml final volume. Add 18. 5 g CsCI and st1r until dissolved. 4 . Add 0.5 mgl ml Hoechst 33258 dye dropwise with m1x1ng to approximately 1 IJg dye per IJg DNA. Stop the addition if the solut1on becomes cloudy since this can result in the precipitation of the DNA . 5. Adjust the density of the solution to a refractive index of 1.3950 at 25 °C by adding either more CsCI or more 10/ 1 TE buffer. 6. Centrifuge the mixture at 40 000 r.p.m . in the Beckmann 50.;1 or Ti60 rotor for 48 hat 40 000 r.p .m. at 25 °C to establish the density gradient. 7. Visualize the blue-fluorescent DNA bands with 3000 A 0 UV 1llum1nation and recover the minor upper band. The lower band contains the undigested kDNA networks and released min1circle DNA . 8 . Re-adjust the refractive 1ndex of the upper band to 1.3950- 1. 3935, and centrifuge 6.5 ml per tube in the Beckmann 50 rotor at 39 000 r.p.m . for 48 hat 25 ° C. Again recover the upper band. This rotor provides a better separation and the second centrifugation completely removes any contaminating minicircle or kDNA. 9 . Remove the Hoechst dye by extraction with an equal vol of propan-2-ol. 10. D1alyse the maxic~rcle DNA against 10/ 1 TE buffer and concentrate the DNA using sec-butanol (see Protocol 3, step 1 2) 11 . Recover the DNA by ethanol precipitation (see Protocol3, steps 1 Ei and 16).

4. Isolation of the kinetoplast-mitochondrion Kinetoplastids contain a single complex mitochondrion per cell. The pornon of the mitochondrion that contains the kDNA nucleoid body is called the kinetoplast. Most kinetoplastid cells are very resistant 10 shear forces in isotonic media. However, in hypotonic media, most swell and consequently can be ruptured by shear forces, thereby releasing the swollen kinetoplastmitochondrion (48). Due to the multilobular nature of the single mitochondrion, isolation of the entire organelle is probably not possible. When the swollen cell is ruptured, the single mitochondrion breaks at the narrow portion adjacent to the nucleus. After resealing, however, the mitochondrion portion contaming the kDNA can be isolated by its relatively high buoyant density ( 1.2 g/ ml) (49}. Often the kinetoplast remains associated with the basal body of the flagellum, indicating some type of attachment, which is also apparent in vtvo. The kinetoplast portion shrinks to a cretenated disc upon addition of 0.25 M sucrose a nd is resistant to exogenous DNase I. Digestion with DNase I to remove nuclear DNA followed by density gradient centrifugation are then used to recover the kinetoplast-mitochondrion fraction as described in Proroco/6. The use of

81

3: RNA editing in mitochondria

m

_J (.)

m

m

m

m

1::1

a

el

0

_J (.)

_J (.)

u

_J

(.)

_J

_J

u

u

!D

u

u

m

~

~

~

m

-' u

u

_J

-' u

m 0

N

_J

_J

u

(.)

Figure 2. Schizodeme analysis. Acrylamide gradient gel profiles of EcoR 1 digests of kDNA from several T. cruzi CL strain cultures and clones. I, CL cells kept tn culture for 2 years (1978-80) and harvested in the stationary phase: II, CL cells harvested in log phase; Ill. CL cells kept at - 70 °C for 2 years I 1978-80); IV, CL cells kept in mice for 2 years ( 1978 -801 and grown for 10 passages in culture; CL 10, 1 1, 14, 12, 16. 18, and 20, are clones from the parental CL strain. Zymodeme groups IB, C) are given in parentheses Note the presencn of two schizodemes in the stock CL stram. Reprinted from ref. 26 w1th perm1ssion.

Renografin gradients gives better results than sucrose or Percell gradients, and flotation is better than sedimentation from the top. However mitochondria from Percoll gradients are more active in transcription assays (50) (see Protocol II) than are mitochondria from Renografin gradienrs. Protocol 6 . Isolation of kinetoplast-mitochondrion fraction Equipment and reagents e STE buffer (85.6g sucrose, 100ml of 0.2 M Tris- HCI, pH 7.9, 10 ml of 0.2 M EDTA, H20 to 1 litre)

• DTE buffer; a 1: 100 dilution of 10/ 1 TE buffer (see Protocol 3)

Continued

82

Larry Simpson. Agda ,\1. Simpson. and Beat Blum Protocol 6. Continued e STM buffer 185.6 g sucrose. 100 ml of 0 . 2 M Tns-HCI pH 7. 9, 3 ml of 1 0 M MgCI,. H2 0 to 1 litre)

• 1.75 M sucrose • SET buffer, DAPI, and UV microscope as in Protocol 3

• 60 ml plastic synnge f1rted with a 26 gauge needle and a p1ston dnven by compressed air at 100 p.s.i, or a d1S· pensing pressure vessel (see Protocol31 fitted With a 26-gauge needle • Beckman ultracentrifuge and SW28 tubes, Sorvall RC-5 centnfuge and SS34 or GS3 rotors, plus appropriate tubes (or equivalents)

• L tarenrolse cells harvested at late log-phase (50-150 x 106 ceJis,mll from e1ther roller cultures ( 1 litre) or aerated stirred cultures ( 13 litre) as described 1n Protocol 2

• DNase I (Sigma, electrophoretically pure), 2 mg/ml in 10 mm Tris-HCI , pH 7.4, 10 mM CaCI,. 50% glycerol

For Renografin gradients • 76% Renograf1n (Squibb Pharmaceutical Co .. 66% diatrizoate meglumme and 10% d1atrizoate sod1uml • 76% RSE buffer (8.56 g sucrose. 50 )II of 0 .2 M EDTA. 76% Renografin to 100mll • 20% RSTE buffer (26.3 ml of 76% Renografln, 8 56 g sucrose. 10 ml of 0 .2 M Tns - HCI, pH 7 9 , 50111 of 0.2 M EOTA , H, O to 100 mil. adJust the density to 1 14 g/ml • 35 % RSTE buffer (46. 1 ml of 76%

Renografin. 8 . 56 g sucrose, 10 ml of 0.2 M Tris- HCI. pH 7. 9, 50 JJI of 0. 2 M EDTA, H 2 0 to 100 ml); adjust the density to 1 26 g 1ml • 20-36% RSE gradients. Layer 16 ml of 35% RSTE buffer in a senes of SW28 rubes; gently overlay with 16 ml of 20% RSTE . Freeze the tubes at - 20 °C. Thaw the tubes overnight at 4 °C before using. The gradient is created by the freezing and thawing. The advantage 1s that many tubes can be easily prepared and stored frozen until use

For Perco/1 gradients • 93 % Percoll (Pharmac1a) In 0 .25 M sucrose, 10mM Tris-HCI, pH 7 . 9, 0.1 mM EOTA, 0.01 mM 2-mercaptoethanol, s1milar soluuons conta1nmg 52. 5% Percoll and 16% Percoll

• 16- 52. 5% Percoll gradients formed as for Renografin gradients (see above) usmg 16% Percoll and 52.5% Percoll solut1ons

Method 1 . Wash the cells by resuspending them m 50-100 vol of ice-cold SET buffer and centrifuging at 4000 g for 1 0 min at 5 °C. Repeat this wash1ng Alternatively, add SET to the cell filtrate when the Millipore Pellicon system (see Protocol 2) is used. 2 . Thoroughly resuspend the cells in X vol. of cold DTE buffer and leave them on ice. X is calculated by dividing the total cell number by the factor, 1. 2 x 10 9 3 . Mon1tor the cells for swelling by phase- contrast microscopy. At 1000 x magnification the swollen kinetoplast-mitochondrion is readily seen. It often 'pops ' out of the cell and remains attached by the flagellum. Be careful not to induce rupture by shearing when applying the coverslip. If insufficient swelling has occurred, add more DTE buffer; 5-1 0 min on 1ce is generally sufficient for complete swelling.

Continued 83

3: RNA editing in mitochondria

Protocol 6 . Continued 4. Break the cells by passage through a 26-gauge needle. For 10-1 00 ml of cell suspension, use a 60 ml synnge with air-driven piston. For 100 ml-31itre volumes, use the dispensing pressure vessel. When the needle becomes clogged, close the shut-off valve and replace the needle. Monitor the extent of breakage by phase -contrast microscopy at 400 x magnification. There should be no more than one intact cell every :3-4 fields. If there are more mtact cells. they will contaminate the final fraction , as VIsualized by the presence of cytosolic rRNA•. 5. Add 0.125 vol. of 1.75 M sucrose to the lysate directly after rupture. This causes the intact kinetoplast-mitochondrion to shrink back into a highly refractile cretenated disc. 6. Centrifuge the lysate at 5 ° C for 10 min in the SS34 rotor at 11 500 r.p.m. or in the GS3 rotor for 15 min at 9000 r.p .m. 7 . Aspirate the supernatant carefully since the pellet is loosely pacl<ed. Resuspend the pellet in STM buffer (50 ml for each 2 litres of the lysate in step 41. 8 . Add 0.005 vol. of 2 mg/ml DNase I and incubate for 1 h on ice to di!~est the nuclear DNA which will otherwise cause aggregation of the material in the density gradients (steps 13-1 5). 9. Add an equal vol. of STE buffer to stop the reaction 10. Centrifuge the solution in the SS34 rotor for 10 mm at 1 1 500 r.p.m . or m the GS3 rotor for 15 min at 9000 r.p.m. at 5 °C. 11 . Aspirate the supernatant; the pellet should be well packed if the DNase digestion was successful. Now proceed to separate the kinetoplast-mitochondrion fraction using either Renografin or Percoll gradients.

A. For Renografin gradients 1. Add cold 76% RSE buffer to the pellets (4 ml of 76% RSE per litre of original culture). Vortex well. 2. Layer 4 -5 ml of the mixture beneath each 20-35% RSE gradient b us1ng a 1 2 ml syringe with polyethylene tubing attached to an 18-gauge needle. If the first drop of lysate floats, add more 76% RSE buffer to the lysate until the lysate remains at bottom of gradient. 3 . Centrifuge the gradients for 2 h at 24 000 r.p.m. at 5 °C in a Beckman SW28 rotor. 4. V1sualize the kinetoplast-mitochondrion band just above the 76%-35% RSE interface by side illumination with a UV lamp. Puncture the side of the tube and remove the band using a syringe and needle. 5. D1lute the m1xture with 2 vol. of STE buffer and centrifuge the prepa~at1on in an SS34 rotor for 15 min at 11 500 r.p.m. at 5 ° C. 6. Check the purity of the preparation at this point, if desired; m the next step, the use of STM buffer will cause aggregation of the mitochondria. To check the purity, resuspend a sample of the pellet m STE buffEn and

Continued 84

Larry Simpson, Agda M. Simpson, and Beat Blum Pro t ocol 6 .

Continued

check the purity by phase microscopy at 1000 x magnification. Visualize the kDNA by staining with 1 IJI DAPI and observing by phase-contrast and UV microscopy. 7 . Wash the pellet by resuspending it in 50 vol. of STE buffer and centrifuging as in step 5A. Finally wash the pellet with STM buffer prior to kRNA isolation. 8 . Resuspend the mitochondrial pellet in an appropnate medium (see Protocols 7, 9 , 7 7, 75). Use it 1m mediately or store 1t frozen at -70 °C in small aliquots. B. For Perea// gradients 1. Resuspend the pellet in 93% Percoll (use 4 ml of Percoll for the pellet from a 1 litre culture). 2 . Layer the mixture under 16- 52. 5% Percoll gradient (use 2 gradients for material from 1 litre culture). Centrifuge for 45 min at 24 000 r.p .m. in a Beckman SW28 rotor at 5 °C. 3 . Locate and remove the kinetoplast band (see step 4A). 4 . Dilute the kinetoplast band with 200 ml of STE buffer. Recover kinetoplast· mitochondrion fraction by centrifugation at 12 000 g tor 30 min at 5 °C. Wash the preparation once in STE buffer and resuspend it in STE buffer. 5 . Store the fraction as for Renografin gradients (step SA). • A quantitative estimate of the contamination can be obtained by measuring the relative amount of cytosofic rRNA in the final preparation of kRNA (see Protocol ll. The cell lysate can be used to prepare cytosohc rRNA substrate for Protocol 12. b The RSE gradtent procedure is the rate-limiting step in large-scale preparations. The lysate from up to two litres of culture can be loaded on each Renografin gradient and six gradients can be run 1n the SW28 rotor.

Figure 3 illustrates the use of Renografin gradients to prepare the kinetoplastmitochondrion fraction from L. tarenrolae.

5. Isolation of transcripts from the mitochondrial

genome 5.1 Introduction and strategy Isolation of intact RNA (kRNA) from the purified kinetoplast-mitochondrion fraction (prepared as in Protocol 6) is easily accomplished due to the lack of nuclease activity in this fraction (49). The major steady-state components of the kRNA are the 9S and 12S mitochondrial rRNAs and tRNAs (51). All the mitochondrial tRNAs appear to be nuclear-encoded and must be transponed into the mitochondrion by a mechanism as yet undetermined (52, 53). Unedited transcripts of maxicircle structural genes can be detected by Nonhern blot 85

3: RNA editing in mitochondria

A

0 Q

4

::E

Q.

u

2

8

Q

4

::E Q. u

2

E: c

e

..... 02 0

~

c Ci

~ 01

0

d:

76% 35%

RG

RG

Fr ocloon (I ml)

20% RG

Figure 3. Renografin isopycnic density gradient fractionation of the kinetoplast-mltochondroon fract1on from L tarenrolae. Gradients were fractionated and fractions washed with STE buffer !Protoco/6) . Aliquots were assayed for (A) TUTase (Protocol 12l and run-on transcnpt1on (Protocol9l act1v1t1es; (Bl RNA ligase activity (Protoco113l (Cl adenylate kmase activity and prote1n. Adenylate k1nase is a marker enzyme for the mitochondrial inner membrane space. RG, Renografm. Repnnted from ref. 50 with permission.

hybridizaton using specific oligonucleotide probes. Edited transcripts can be detected using genomic DNA probes if the editing is not extensive. However. mature pan-edited mRNAs, such as the L. caremo/ae RPSI2 RNA (54), can be detected only by knowledge of the edited sequence. In this case. primar transcripts containing pre-edited regions can be monitored using genomsl DNA probes. Funhermore, one can take advantage of the 3 ' _. 5' polarit'. of editing to detect partially-edited mRNAs which still contain genomic 5 sequences, b) selective PCR amplification using an oligo(dT) 3' primer for the first strand synthesis and a genomic 5' primer for the subsequent PCR 86

Larry Simpson. Agda ,\f. Simpson. and Beat Blum varie~ from gene to gene (56). Partially-edited mRNAs are usuall~ le~s abundant than edited or pre-edited RNAs and can only by detected b) selective PCR amplification in L. tarenrolae. The gRNA transcripts which are involved in RNA editing co-migrate with tRNA in agarose gels and migrate ahead of tRNA in polyacrylamide gels (52). The 3 ' oligo(U)-tails of gRNAs vary in length from 5-28 nt and are responsible for the chracteristic family of electrophoretic bands each differing b} a single nucleotide (57). The abundance of gRNAs is relatively low compared to mitochondrial tRNAs and so the} can not be visualized in gels stamed \\ith ethidium bromide unless 4-5 ~g of kRNA is used per lane.

(5-t. 55). The ratio of pre-edited transcripts to mature edited mRNA

5.2 Isolation of kinetoplast RNA Protocol 7 describes the isolation of kRNA from the kinetoplast-mitochondrion fraction prepared by Renografin gradient centrifugation (see Protocol 6). Protocol 7 . Isolation of kinetoplast RNA Equipment and reagents fugat1on (see Protocol 6) and w1th the mitochondna from 1-21itres of origmal cell culture resuspended in 5-1 0 ml of TMN buller; may be frozen at - 70 °C until use

e TMN buffer 110 mM Tns-HCI, pH 7 5 . 10 mM MgCI2 5 mM NaCil • 2 mg·ml DNase I (RNase·free: BRL. 1180475Al • Phenol :chloroform ( 1 1. v lv) • 1 0 mg/ml ethidium bromide • Kinetoplast ·mitochondrion fraction: prepared by Renografin gradient centri·

• 1% agarose gel 1n TBE buffer !Volume I, Chapter 1, Protocol 6 or Volume I, Chapter 4, Protocol 12) e 10% SDS

Method 1 . Quickly thaw the kmetoplast-mitochondrion fraction (if necessary) Add 0.01 vol. of 10% SDS and incubate the mixture on 1ce for 5 mm. 2 . Extract the mixture by vonexing with an equal vol. of phenol:chloroform ( 1: 1) as described in Protocol 3, step 13. Remove the aqueous (upper) phase to a fresh tube.

3. Re-extract the 1nterface with one vol. of water and pool this with the upper phase.

4 . Ethanol-precipitate the nucle1c acids, (see Protocol 3, steps 1 5 and 16). 5. Resuspend the nucle1c ac1ds m TMN buffer usmg 1 ml TMN per 2- 31itres of origmal cell culture

6 . Add 0.005 vol. of 2 mglml DNase I and d1gest the m1xture for 30 min at 37 °C 7 . Extract the solution with phenol·chloroform and precipitate the kANA w1th ethanol as descnbed in steps 2-4.

Continued

87

3: RNA editing in mitochondria Protocol 7 . Continued 8 . Wash the pellet in 70% ethanol. Resuspend it in water ( 1 ml per 41itres of original culture yields approximately 2 ~g RNA / ~1) .

9 . Monitor the purity of the RNA by electrophoresing 2 - 5 ~g in a 1.5% agarose gel in TBE buff er, and stain1ng the gel with ethidium bromide (0. 5 ~g/ml) . Any contaminating cytosolic rRNA will be apparent as three bands migrating above the mitochondrial 125 rRNA band (the cytosolic large rRNA has a break and gives rise to two bands in gel electrophoresis) . Also visible should be the 9S rRNA and the tRNA bands (51). 10. Store the kANA in aliquots at -20 °C.

5. 3 Northern blot anallysis of kRNA Northern blot analysis is described in Volume I, Chapter 2, Section 3.6 and Volume I, Chapter 4, Section 7.5. Electrophorese the kRNA (see Protocol 7) in a formaldehyde-agarose gel and blot the gel onto a nylon fi lter. Hybridize the filter with an appropriate radiolabelled probe complementary to the kRNA sequences under investigation. For internal size standards, strip and reprobe the kRNA blot with a nicktranslated pLt120 probe, which represents a 6.6 kbp fragment of the maxicircle DNA containing the 9S and 12S r RNA genes, and the ND7, COlli, and Cyt 6 genes (58). The resulting bands in the autoradiograph should be 320 nt, 610 nt, 1100 nt, and 1200 nt in size.

5 .4 Hybrid selection of guide RNAs A hybrid selection procedure can be employed (57) to isolate specific gRNA:. The method makes use of synthetic oligonucleotides which contain a reactive amino group at the 5 '-end. The amino group is covalently reacted with the hydroxyl groups of agarose in the presence of a strong reducing agent. This step immobilizes the oligonudeotides to the agarose which can then be used for affinity purification of specific RNA species by column or batch procedure~. Protoco/8 is a modification of the filter hybridization method of Wood eta/ (59). A stringent wash removes non-specifically bound RNAs before the selected RNAs are eluted by using a low-salt buffer at an elevated temperature. Prot ocol 8 . Hybrid selection of guide RNAs

Equipment and reagents • SynthetiC DNA oligonucleotide: 100 l)g; 20-30 nt complementary to the gRNA to be isolated ; at the last step of the synthes1s, couple the reagent, Amino link 2 (Applied Biosystems) to the 5 '-end, following the supplier's instnJctions

• kANA from 5 lit res of a log·phase Leishmania culture at a cell density of 0. 7- 1.5 x 1 0 8 C'ells/ml for the hybnd selection of each gRNA (see Protocol 7) e 10% SDS

Continued

88

Larry Simpson, Agda M. Simpson, and Beat Blum Protocol 8 . Continued e lmmunoPure Ag/Ab Immobilization kit (Pierce 44890) together With immobiliza· tion buffer as provided by the manu· facturer

• 6 X SSe buffer and 1 X SSC buffer (prepared using 20 X SSe buffer Stock)

m1crocon·

• 0.1 X sse buffer, 0.1% SDS (prepared using 20 )(sse buffer stock and 10% SDSI

• 50 ml centrifuge tubes with plug seal caps (Corning 25331-50)

• Hybndization buffer (1.0 M Nael, 0.166 M Hepes-KOH. pH 7.5, 1 mM EDTA, 0 .1 o/o SDS)

• Centricon 1 0 centrifugal centrater (Amicon 4205)

• Rotating platform stirrer

• Wash buffer (10 mM Tris-HCI, pH 8.0 , 1 mM EDTA)

• 20 x SSC buffer stock (3.0 M NaCI, 0.3 M trisodium citrate, pH 7.0 with NaOH) • 5.0 M tetramethylammonium chloride stock solution (Fischer # 04640-5001; adjust the density to a refractive index of 1.421 at 25 °C • 1.0 M Tris - HCI, adjust the pH to 8 .0 using HCI e 0.5 M EDTA, pH 8 .0 • TMA buffer (3. 0 M tetramethylammonium chloride, 50 mM Tris- HCI, pH 8.0. 2 mM EDTA, 1% SDSI

• 2 ml agarose beads together with the appropriate chromatography column (as provided by the lmmunoPure Ag/Ab immobilization kit, Pierce 44890) • Carrier nucleic acid (5'-amino oligonucleotide, not complementary to the gRNA to be hybrid selected); 5'-amino linked oligonucleotides are convenient carriers because they do not Interfere with the 5' labelling reaction which follows the hybrid selection step; circular DNA molecules or glycogen may work as well

Method 1 . Attach 100 1J9 of the aminolink 2-oligonucleot ide to 2 ml of agarose beads in 2 ml of the recommended immobilization buffer.

2 . At the end of t he required incubat ion period pack the agarose into the column and wash it twice wit h 5 ml of wash buffer and store the washed column at 4 °C until required. 3. For hybrid selection of gRNA, equilibrate the column in hybridization buffer.

4 . Load approximately 500 IJg of kANA in 1 ml hybridization buffer onto t he column. 5 . Seal the column with the cap and stopper provided; incubate it for 18 h at 37 °C. 6 . Drain the column and add 5 ml of ice-cold 6 X sse buffer. 7 . Repeat step 6 twice, then incubate the column at 4 °C for 2 h with gentle agitat ion by placing the column into a 50 ml centrifuge tube and rotating it horizontally on a rotating platform stirrer which is set to 50 r.p.m . 8 . Drain the column and equilibrate it with 5 ml of TMA at room temperature. 9 . Repeat step 8 and place the sealed column into a 50 ml centrifuge tube. Seal the centrifuge tube and place it into a 37 °C water bath for 30 min. 10. Drain the column, then add 5 ml TMA buffer (preheated at 60 °CI. Again seal the column into a 50 ml centrifuge tube and place into a 60 °C water bath for 30 min•.

Continued 89

3. Rt\A editing in mitochondrw Protocol

8 . Continued

11. Wash the column successively With l O ml of TMA buffer at room temperature, 10 ml of iCe·COid 6 X SSC buffer, and 10 ml Of ICe-cold 1 X SSC buffer. 12. Elute the gRNA at 70 °C for 20 min USing 2 ml of 0 . 1 X sse buffer, 0.1 % SDS. preheated to 70 °C. This is done by placing the column into a sealed 50 ml centrifuge tube and immers1ng It into a 70 ° C water bathb. Collect the column eluate. Repeat th1s twice With fresh buffer.

13. Add 2 1.19 of carrier nucle1c ac1d . Concentrate the pooled eluate w1th the Centricon 1 0 centrifugal microconcentrator following the manufacturer' s instructions.

14. Store the hybnd-selected gRNA at - 20 °C . • Note that the exact wash1ng temperature mu51 be vaned accordmg to the size of the hybrid (591. H1ghly stnngent washong occurs at T_ - 4 °C. The T.., of a hybnd m TMA buffer is solely dependent on its length and can be calculated from ref 59 • Do not heat the columns containing the agarose beads to more than 70 ° C. The agarose could melt above this temperature

5.5 PCR amplification of partially-edited mRNAs and gRNA:mRNA chimaeric molecules Partially-edited RNAs can be amplified by the use of an oligo(dT) 3 ' primtr complementary to the oligo(U)-tail of the gRNA. for the reverse transcript ·~e step (60). This is followed by the addition of a 5' primer consisting of a genomic sequence complementary to the unedited 5' sequence of the mRNA and "aq polymerase for the PCR amplification cycles. In a modified procedure, it is possible to select for partially-edited molecules covering a limited reg10n of the mRNA by choosing the appropriate set of primers (55, 61). In this ca e, the 3 primer should cover the edited sequence while the 5' primer sht. ld coYer the upstream pre-edited sequence. PCR amplification selects for R ~A molecules partially edited in the sequence between the two primers. Tr 1s approach allows for fine analysis of partially-edited molecules at single s1tes. In vivo chimaeric gRNA:mR A molecules have been detected in kR .\ by Northern blot hybridization and by PCR amplification using a 3 · primer complementary to mRNA downstream of a PER (16). The polymerization of eDNA using reverse transcriptase is followed by the addition of a 5' primer with the 5' terminal sequence from the corresponding gRNA. PCR amplification reveals the presence of chimaeric molecules. The existence o f uch chimaeras is indicative for the trans-esterification model. However, they could also arise as by-products of a cleavage-ligation process. Detailed procedures for PCR will be found in Volume I, Chapter 2, Section 3.4 and Volume I, Chapter 3, Section 2.2.3 and in ref. 62. PCR produ,·ts both from partially-edited and chimaeric molecules may be checked b> gel electrophore:;is and their nucleotide sequence~ determined. Figure 4 sho· s partially-edited sequences in the maAicircle G-rich region 6 of L . rarentola~.

90

Lorry Simpson. Agdo M. Simpson, and Beat Blum

5.6' Synthesis of gRNAs by transcription in vitro Using bactenophage promoters and purified phage RNA polymerase, gRNAs be transcribed in vitro. For instance, a T7 phage promoter sequence can be incorporated mto the 5' PCR primer for gRNA amplification (see Section 5.5). The amplified DNA is then transcribed using T7 RNA polymerase. The transcription products will have heterogenous 3' termini due both to the propensit) of the polymerase to add an extra non-encoded A or C and to premature termination (63), and so the desired species must be purified by gel electrophoresis. Such transcripts have been used to study the formation in vitro of gRNA:mRNA chimaeric molecules in a mitochondrial extract (18-20). Procedures for the transcription of RNA in vitro using phage promoters and polymerases will be found in Volume I, Chapter I (Protocol 4) and Chatpter 2, this volume (Protocol 4). 01 ay

6. Run-on transcription in isolated kinetoplast

mitochondria Kinetoplast mitochondria isolated by the procedure described in Protocol 6 are active in run-on transcription \\'hereby RNA chains initiated in vivo are completed in vitro. As explained in Section 4, mitochondria prepared in Pero:oll gradients are generally more active in run-on transcription than those prepared usmg Renografin gradients . Nevertheless, mitochondria prepared by the latter procedures are adequate for studying run-on transcription. Run-on transcription may also be studied in mitochondrial extracts, prepared as described in Section 8. Protocol 9 describes run-on transcription using intact mitochondria. Using this method, the rate of incorporation of [ a-32P] GTP increases for J0-15 min and then decreases.

r-f rotocol 9. Run-on transcription in isolated mitochondria 1

I

Heagents t1 5 x STE buffer !see Proroco/6 1 • Mitochondnal fractiOn ISolated by Renogratin or Percell gradients as descnbed 1n Protocol 6 , resuspended at 2- 3 (JQ/IJI in STE buffer for immediate use or stored frozen on STE w1th 8% DMSO at - 70 °C; run-on transcription actiVItY is stable for several weeks 1n frozen mitochondria •• 10 mM ATP •• 10 mM CTP

• [o- 2P]GTP (New England Nuclear; 3000 Co mmol. 10 mCo mil • 30 mM potassium phosphate, pH 6.8

• 2 M KCI

• O 25M Hepes-KOH, pH 7.6 • 0.25 M 2-mercaptoethanol

• DE81 filter d1scs (Whatman, 2.5cm diameter) • 0 . 5 M sod1um phosphate buffer, pH 6 8. 0.5% sodoum pyrophosphate

•• 10mM UTP

Continued

91

A DNA: RNA: Sl te: Clone: 1

GG TTGTTTGTTHTG 0 GAA CTTA G CTTG A A G GAATTTTTTGG GG GG A GA GCCAGGAG AAAG A TTTCACG GAATTGTTT!CGT AIJUUWU<JGuCUUUIJ.AJG WG UGuuuCuuuuuuGAAUC AuuuC C UGuwAuA GwGAA GGuCGuCGuuuuAuuGAuuuCCCAGGAGuAAAGuAWAJIJCACGuuuuuuuuuGAA UG CGU 26 25 2' 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2

21

G CTTG A A G GAATTTTTTGC GG GG A GA GCCAGGAG AMG A 1 TTCACG GMTTG1TT1CGT CM CTTA GG TTGTTTGTTTTTG GAA. CTTA G CTTG A A G GMTTTTTTGG GG GG A GA GCCAGGAG AMG A TTTCACG GAA~G CGf TTGTTTGHTTTG G GC A GA GCCAGGAG MAG A lTTCACG t.nUitGM TG· ,.,_ (Gt GAA CTIA G CTTG A A G GAATTTTTTGG GG CG A TTGTTTGTTTTTG G GG GAA CTTA G CTTG A A G GMTTTTTTGG GG GG A GA GCCAGGAG AMG A TTTCACCtittt~~t'ti)M' tC., t(;t A TTGTTTGTTTTTG GG GM CTTA G CTTG A A C GAATTTTTTGG GG GC A A GA GCCAGGA~AM~~A.tUTTCACGtttttt(t~GAA JG CGf TTGTTTGTTTTTG G GG A TTGTTTGTTTTTG G GAA CTTA G CTTG A A G GAATTTTTTGG$.G.~~~~~-H:~~H.QltnCiC:~A~~CJ,A#~~.A~'tUTCACCt tttt~tttGM TC cC:'r GG GAA C A GtC TG tA AttG GAA: ti?t{"::GtGCtGCthtGtt~Gi'ftGCCA~~GtM.(~iAttTTTCACCtttttttttCM TG TTGTTTG_TTTTG G CGr A GG GAA CTTA G CTTG A A GttGAA ;.<8if/CGtGGtGGttttAt'(~A'tttGCCA,GGAGtMAG\A{tllTCACGttttttttlCM JC cor' A TTGTTTGT I TTTG G GG GAA CTTA TTGTTTGTTTTTG G C!L_C A A Ct~W .,i}'(} cat!1~ : ".,.~qJ: lGtttGt tt(~~CMtC AtttC C·rctttAtA CttGAA. ")' GGtGGtGCttttAtltAfttCCC:AGCACtWG.tAtti'TTCACGtttttttt tGAA TG C~T Attt tt ttGtGt tl tlTG.)TG TCtttGttttttGM.£..1AtttC ( TGtttAtA CttCM· ,,., CGtGGtGQttttAttCAtttGCC~CGAGtAMGtAttTTTCACGtttttttttGAA TC CGT Attt ttttGtGU~tHG. JTG'

22 23

A A

2

A

s• 10 11 12 13 14 IS 16

,,

18..

24

25

26

CG TTGTTTCTTTTTG G GM CTIA G CTTG A A C GAATTTTTTGG GG GG A GA GCCAGGAG AMG A TTTCACC GAATTGTTTTCGT fttCtCttttTTG ''lTG TGtt!CtttttJGAAtC AtttG C 'TG.t ttAtA GttCM GCtCCtCGl\~t.AttGAtttGCCAGG~G_AAA.GtAUTT TCACGtttttttttGAA TG CGT :~ftttlt).~tCtt~tTWJTif'' TCtttCttt.tttGAAtt .A\ttC. C."lG.t ttAtA GttGAA,: · · GGtCiltCGtttt.AttGAUtGC,._GGMtAMG~AtiTTTCACGttttttlttGM JG CGT ttA G C tttTTG TTG TT G ttrCt'tttttQ~MC AtttG C.JGtttAtA GttCM GGtCGtCCqttAttCAtttGCCAGCAGtAMGtAttTTTCACCtttttttttGAA. TG AGAtttt tGttG TTGTTTGTTTTTG G GAA C TA tC C''·TC~ttAtA GttGAA ..,:::: ·,::. G~tGGtl;Ctttt~t~CAttiCCCAGCAGtlliGtAttTTTCACGtttttt~UGAA TQ

Ident tc:al c lones: 6 , 7, 8 , 9 1<1, 20

CCT CGT

8 DNA:

TC

G

A GTlTG

GuuuAuG

RNA:

Site:

55

54

UG

· 52

CTHTG

51

TTATTTTA

GIJUUI.AJUA

uuC

50

'9

G

GG

TA A

MG CCACGA

ACCTAGTTCCGG AATCGACG

G

A ATTTGCAA

AG AAG

AA A

AG

UAuuuuuGuuuGGuuUJAuAuuuAAGUCCACGA uuACCUAGUUCCGGuAAUCGACGwGuuuAuA UGCAA uAuGuuAuAUCuuuAAuAuuwAuG

48

47

46

45

44 4l

42

41

40

l9 l8 l7 36

35

·

31

lO

29 28 27

Clone:

17 18

G

IG

A GTTTG

CTTTTG

TTATTTTA

rc

A GlTTG

CTHTG

HATTTTA

TG

G G

lTATTTTA TA A

19

G

20 21

Gtttttt t$1G

A G JTGtttt~ A G TG

GttttttfiT~

~

22 2l 24

Gttttttti[G

AG

G G

TG

G

TG

A GTTTG

CTTTTG

TG

A GTTTG

CTTTTG

25 26

TG

Q lG

Q

rc

A G !G A G TG

c

TA

G G

GG GG

TA A

MG CCACGA

ACCTAGlTCCGG MTCGACG ACCTAGTTCCGG MTCGACG

G G

A ATTTGCM

AG AAG

MA

AG

A ATTTGCM A G A~AtCtttMtAt tttAtG G GG TA A AAG CCACGA ACCTAGT TCCGGtM TCGACGttGt t tAtA TGCM tAtGttAtAtGU.t.M~AttttA.t¢ Gt t tGCt t t TA tAt t tMG tCCACGAlll:CCTAGTTCCGGtM TCGACG t tG t t tA tG TGCM tAtGttAtAtGtttMtAttU~\GTA A

MG CCACGA

Gt t tGGtttf.AtAt t tMGtCCACGA t tACCTAGTTCCCG tM TCGACCt tC t t tAt A TGCM tAtCttAtAtGtttMtAttttAtG

!A

A

Gt ttGCtt tfAtAtttMGtCCAtGA:, ttACCTACTTCCCC AATCGACC

c

A ATTTGCM

G

TA

A

Gtt tGGtt tTAtAt t tMGtCCACGA!ll..:.CCTAGTTCCGG MTCGACG

!i

TA

"

Gt t tGGt t tTAtAt t tMCtCCACGA!ll:.CCTAGTTCCGG M TCGACG

G G

A ATTTGCM A C A A G MA AG AU TGCAAttA\Ct tAtAtCtt.\ AAtAq t tAt

TTATTTTA

G

GG

TA A

MG CCACGA

ACCTAGTTCCGG AATCGACG

c

A A TCCM

TT.t.TTTTA

G

GG

TA A

MG CCACGA

ACCTAGTTCCGG

AG AAG

AA A

AG

a

A C tA At ttGttttAtAtAtAG

MTCC~UBIICtt~AIG~~tAAGAttttAtG!AIMI!l

Figure 4 . Partially edited max1circle G nch reg1on 6 I = RPS 121 eDNA sequences from L tarentolae. kANA was amphf1ed usmg a 3 ' ohgo(T) pnmer and a 5' genom1c pnmer The PCR products were cloned and the clones sequenced Sequences are aligned to show the 3 - 5 ' progress1on of expected editing even ts. Unexpected editing events are underlined. Pattern 5 was seen 1n 5 clones , pouorn 18 was soon In 3 clones. The unedited domam-connection sequences are shaded . fA) 3 ' terminal port1ons of the eDNA sequences. fBI 5' terminal port1ons of the sequence:.. Clones 1- 16 are not shown smce their sequences, like clone 17. are unedited in th1s reg1on. Reprinted from ref. 54 With perm1ssion.

3: RNA editing in mitochondria

Protocol 9 . Continued Method 1. In a microcentrifuge tube on ice mix: • 30 mM potassium phosphate, pH 6. 8

• 2 M KCI

5tJI 1.51JI

e Q.25 M Hepes-KOH, pH 7 .6

1 tJI

• 0.25 M 2-mercaptoethanol

1 IJI

2.5tJI

• 5 x STE buffer [a- 32P]GTP

e

1 tJI

• 10mM ATP

5tJI

e 1QmM CTP

StJI

• 10mM UTP

51JI

• mitochondrial fraction

10tJI

to 50 IJI final volume • H2 0 2. Incubate the mixture for 1 5 min at 27 °C. 3. Remove 10-20 IJI aliquots and spot them onto DE81 filter discs. 4. Dry the discs and then wash them for 30 min in 100 ml of 0. 5 M potassium phosphate buffer, pH 6.8, 0.5% sodium pyrophosphate. Repeat the washing three times. 5 . Wash the discs with 100 ml of ethanol as in step 4 . 6. Dry the discs and count the retained radioactivity in a scintillation counter.

The run-on transcripts, synthesized by the method given in Protocol 9, migrate in agarose gels as a smear, suggesting that they represent nascent RNAs elongated in vitro during the labelling period (50). The labelled RNAs hybrid1ze to all regions of the maxicircle, even to the divergent region which shows very low levels of steady-state transcripts, and to minicircle DNA.

7. Identification of gRNAs by computer-assisted

sequence comparison In the editing of mRNAs, gRNA molecules specify the editing by forming duplexes with the edited mRNA (15). Such duplexes display G-U base pairs in addition to the canonical G-C and A-U base pairing. A search for gRNAs can be carried out with the help of a computer (see also ref. 64). The edited mRNA sequences are compared with the kinetoplast genome (consisting of the maxicircle and the known minicircle DNA sequences) taking into account such non-canonical base pairing. Prior to the computer search, the sequence of all or part of the edited mRNA must be obtained by direct RNA sequencing. Reference 65 gives details of RNA sequencing methods. Protocol 10 describes the computer analysis. 94

Larry Simpson, Agda M. Simpson, and Beat Blum A modified local homology alignment program (BESTFIT, University of Wisconsin Genetics Computer Group package, version 6) is used in the search for gRNA sequences. The scoring matrix is altered so as to score positively for complementary base pairs (including G-U) rather than for matches. Moreover, it is possible to apply different weights for each possible base pair. Protocol10. Identification of gRNA sequences by computer analysis A. Preparation of the files

1. Download the kDNA genomic sequences from the Genbank database. 2. Reformat the files to GCG format. 3 . Create a GCG sequence file with the determined edited mANA sequence (see SEQED, UWGCG) . 4 . Reverse the mANA sequence, using REVERSE (reverse only, UWGCG).

B. Modification of the scoring matrix 5. Edit the scoring matrix SWGAPDNA.CMP (FETCH this file) using an appropriate editor to score for base pairing instead of matches. The following table SWGAPDNA .CMP is an example of weighted scores. A

c

- 0.9

0.01 - 0.9

T

u

0.5

0.5

A

1.0

- 0.9

- 0.9

c

- 0 .9

- 0 .9

G -0.9

- 0 .9

0 .25 - 0.9

G T

-0.9

u

Note: This table does not 'punish' for C-A base pairing; C-A pairs have been found in putative gRNA:mRNA duplexes from C. fasciculata (66).

C. Running the program 6. Type: BESTFIT/DATA 1 = SWGAPDNA .CMP/PAI =0.0 1 7 . Select a high value for gap weight ( * 100 •) and gap weight length ( * 2.00*) to avoid any alignments with gaps.

8 . In order to find the gRNA sequences corresponding to an edited mRNA sequence, it may be necessary to vary the size and borders of the input mRNA sequence (e.g . at the sites of two overlapping gRNAs). Known gRNAs help to define the borders for the search of additional gRNAs. The smaller the size of the gRNA:mRNA duplex, the more difficult it becomes to find the corresponding gRNA (64) . In the case of L. tarentolae, each minicircle encodes a single gRNA located 1 50 nt from the end of the conserved region (22!. The DNA sequence of this region can be tested for the presence of gRNA sequences against known edited mRNA sequences. In the case of T. brucei, each minicircle encodes at least three gRNAs located between 18mer inverted repeats (23). One could locate these conserved repeats and computer search the intervening DNA sequences.

Continued 95

3: RNA editing in mitochondria Protocol 10 . Continued D. Search for edited mRNA 9 . In some cases putat1ve gANA sequences (for instance, as 1nd1cated by the presence of a 3 ' oligo(U)-taill have been found pnor to the 1dentificatiot1 of the corresponding edited mANA (52). In th1s case the method described 1n Protoco/9 can be used to search for the corresponding mANA. However when genom1c kDNA is scanned for the presence of the correspondtn' mANA. the search is complicated by the fact that pre-edited mANA does not yet contain the U residues to be inserted or deleted.

8 . Enzymatic activities in the kinetoplast-mitochondrion

fraction which are involved in RNA editing 8.1 Introduction Several enzymatic activities which are thought to be involved in editing maxic1rcle transcripts have been identified in purified kinetoplast-mitochondrion fractions. These include a terminal uridylyl transferase (TUTase) (50), an RNA ligase (50), a site-specific cryptic RNase (67) and a gRNA:mRNA chimaera-forming act ·vity (18-20) (Figure 3). The TUTase adds U residues to the 3 hydroxyl group of RNA mole.. ules with no apparent sequence specifity. The role the TUTase is thought to be he re-addition of U residues to the 3' oligo(U)-tail of the gRNA which has 1een used as the source of UMP for transfer to the editing site of the mRNA in two successive trans-esterifications. The function of the RNA ligase in the mitochondrion is unclear; in the ongmal enzyme cascade model (15) for RNA editing, a ligase is required for joming together the cleaved mRNA fragments after the addition of UMPs to the 3' hydroxyl group by the TUTase, but there is no requirement for an R'lA ligase in the trans-esterification model (16). A cryptic RNase in the kinetoplast-mitochondrion fraction can be acti\ ted by the addition of heparin or by predigestion of the extract with proteinase K or pronase (67). The authors have suggested that the cleavage activity invol ed in the activation is actually a site-specific hydrolysis catalysed by the same ell!yme which normally trans-esterifies gRNA and the mRNA, and that this hydr 1l> 'i~ is induced by inhibiting or destroying the TUTase. It is also possible that this activity is an enzyme involved in RNA turnover. According to the trans-esterification model of RNA editing (16, 17), urid ne residues are directly transferred from the oligo(U) 3 -tail of the gRNA into the mRNA via a gRNA:mRNA chimaeric intermediate. Such chimaeric molecules have been found in steady-state mitochondrial RNA from L. tarentolae ( 16) and T. brucei (68-70). In L. rarentolae, these in vivo chimaeras generally consist of gRNA covalentl) linked via the 3' oligo(U) sequence to the correspond 1g 96

Larry Simpson. Agda A·f. Simpson, and Beat Blum rnRNA at a normal editing site, \\ith the downstream editing sites being fully edited . Recently, mitochondrial extracts have been prepared from T. brucei (18, 19) which show chimaera-forming activities in vitro. For L. tarentolae the chimaeric molecules synthesized in vitro are similar but distinct from the ones observed in vivo (20), in that attachment also occurs predominantly at editing ~ites, but no ediung could be detected do\\<nstream of the attachment site.

8.2 Preparation of mitochondrial extracts several of the activities involved in mitochondrial RNA editing mentioned in section 8. 1 may be assayed in either intact isolated mitochondria or various mitochondrial exuacts. Protocol I I describes the preparation of detergent 1ysates of kinetoplast mitochondria isolated in Renografin gradients (see Proroco/6). These are the TL. TS, and S-100 extracts. Pr<>tocol 11 . Preparation of Triton X-1 00 lysates

Equipment and reagents • K inetoplast-mitochondrion fract1on isolated by flotation in Renografin dens1ty gradients (see Protocol 6). Resuspend the washed mitochondrial pellet at 5 mg prote1n mlin 20 mM Hepes-KOH, pH 7.5. 0 1 M KCI, 0.2 mM EDTA, 20% glycerol.

It may be stored frozen 1n 200 ~I aliquots at - 70 °C

e 10'" Triton X-100 IP•ercel • Pellet pestle mucer !Kontes Glass Co. ~ 749520, motor driven)

Method

1. If necessary thaw the mitochondrial fraction in 1ce 2 . Add 61Jl of 10% Tnton X-1 00 to 200 fJI of the mitochondrial fraction in a microcentnfuge tube. M1x gently. 3 . Homogenize the m1tochondr a for 1 5 sec at 5 °C usmg the pellet pestle m•xer. Th1s homogenate is the TL extract 4. Centnfuge the TL extract for 30 min at 1 2 000 g at 5 °C m a microcentrifuge (to obtain the TS extract) or for 1 h at 100 000 g at 5 °C (to obtain the S-1 00 extract). In each case, carefully remove and retain the supernatant. 5 . If necessary. store the extracts in aliquots at - 70 °C

~----------------------------------------------------~

8.3 Terminal uridylyl transferase (TUTase) The kinetoplast-mitochondrion fraction isolated from Renografin gradients contains a TUTase. This activity, which adds multiple uridine residues to the 3 ' hydroxyl group of RNA molecule~, can be solubilized by homogenization With 0.31t/o Triton X-100. The solubilized TUTase activity may be assayed b:,: the incorporation of UMP residues from UTP. Since it only requires one

97

3: RNA editing in mitochondria nucleoside triphosphate in addition to UTP, TUTase may be assayed in the absence of the run-on transcription activity, "hich wouJd also result in nucleotiJe incorporation. TUTase activity may be studied in either intact mitochond a or mitochondrial extracts. To assay TL'Tase activity in extracts, sub trate R.'\ \s must be pro\ided b) adding cytoso!ic RNA from L. rarenrolae. This has five sm;.UJ rRNA components (215 nt, 195 nt, 175 nt, 140 nt, and 110 nt) that migrate in po yacrylamide gels between the 9S rRNA and the tRNA regions (51). No substr<~te RNA need be added with intact mitochondria, in which endogenous mitochondnaJ RNAs are labelled with [a-32P] UTP, including the 9S and 12S rRNAs and the gRNAs. The mitochondrial tRNAs do not label well with this enzyme actiVIty. Heparin (5 ~g/ml) inhibits the TUTase activity of extracts, but does not affect the endogenous TUTase activity in isolated intact kinetoplast mitochondna, probably due to the lack of penetration through the mitochondrial membrane. Prorocol 12 describes the assay of TUTase in mitochondrial extracts and the preparation of the cytosolic RNA substrate. Under these conditions, the incorporation of UTP occurs linearly for at least 30 min at 27 To as .ay TUTase in intact mitochondria, Protoco/12 should be modified by omit ,g the addition of the cytosolic RNA substrate.

oc.

Protocol 1 2 . Assay of terminal uridylyl transferase (TUTase) in isolated mitochondrial extracts Equipment and reagents • Cell lysate prepared as m Protocol 6, steps 1- 4 • Mitochondnal extract !TL, TS or 5 · 100, see Protocol 1 7) (2-3 1-19 protein/IJI) e 0.25 M Hepes-KOH, pH 7 .5, 2M KCI, 10 mM ATP, and DE81 d1scs as in Protoco/9 • 60 mM magnes1um acetate • l .OM

on

• 30 mM potass1um phosphate. pH 7 0 • 0.5 M sodium phosphate buffer. pH 6.8 0.5% sodium pyrophosphate with and Without 0. 1% SDS • TMN buffer, 10% SDS, 2 mg/ml DNase I solut1ons as in Protocol 7 • 2.0 M Nc1CI and phenol:chloroform as n Protocol 3 e lOmM GTP

• I a· 32P) UTP (New England Nuclear: 800 Ci mmol 10 IJCi 'IJII

Method A. Preparation of cyrosolic RNA substrate 1. Centrifuge the cell lysate (prepared as per Protocol 6 , steps 1-4) at 16000g for 10min at 5 °C. 2 . Remove the supernatant and add 0 .01 vol. IJf 10% SDS. 3 . Deproteinize with an equal vol. of phenol:chloroform ( 1:1) as described in Protoco/3, step 13. Remove the upper (aqueous) phase to a fresh tube. Re-extract the phenol layer and mterface with one vol. of water and pool it with the first upper phase.

Continued

98

Larry Simpson, Agda M. Simpson. and Beol Blum Protocol 12. Continued 4. Ethanol-prec1p1tate the nucleic ac1ds (see Protocol 3, steps 1 5. 161. 5 . Resuspend the pellet 1n TMN buffer ( 1 ml TMN per 2-3 htres of original cell culture). Add 0.005 vol of 2 mg ml DNase I and incubate the mixture for 30 min at 37 °C 6 . Extract with phenol :chloroform and ethanol prec1p1tate the RNA (steps 4, 5). Wash the pellet three times with 70% ethanol and spinvac dry. 7 . Resuspend the RNA from four lit res of original culture in 1 ml redistilled sterile water (0. 5-1 .2 IJg RNA/IJI) and store it at - 20 °C.

B. TUTase assay 1 . In a microcentrifuge tube on ice. mix: e 1 M OTT

1 IJI

• 30 mM potassium phosphate. pH 7.0 e 10mM GTP

5 IJI 51JI

e 0 .25 M Hepes-KOH. pH 7. 5

51JI

e 2 M KCI

1 51JI

• 60 mM magnesium acetate

5 IJI

e 10 mM A TP

5 IJI

e [a-32P]UTP

11JI

• mitochondrial extract

10 IJI

• cytosohc RNA substrate Iprepared as above) • H2 0

1 Ill to 50 IJI final volume

2 . Incubate the mixture for 40 min at 27 ° C. 3 . Stop the reaction by adding 50 Ill of 0.5 M sodium phosphate buffer. pH 6.8, 0. 5% sodium pyrophosphate, 0.1% SDS . 4 . Remove 80 Ill aliquots onto DE81 filter discs. 5. Process the discs and determme the retained radioactivity as described in Prorocol 9, steps 4-6.

8.4 RNA ligase An RNA ligase activity was first observed in rotal cell e>.1racL~ from T. brucei (71 ). The relationship of the total cell RNA ligase acthity to the mitochondrial activity described in L. tarentolae is not clear. The L. rarentolae RNA ligase acti\ ity co-sediment~ with the kinetoplast-mitochondnon fraction (50). It is solubilized in Triton X-100 and thus can be assayed in mitochondrial extracts. Incubation of cytosolic RNA with mitochondrial TL extract and [ ~- 32 P) UTP under the conditions required for TUTase in the presence of ATP and Mg2 + ions yields a 180 nt labelled RNA product whose relative electrophoretic mobility varies with the gel concentration, a behaviour characteristic of circular RNA molecules. RNA ligase is quantitatively assayed by the addition of [ a- 32 P) pCp to the 3 ' termini of RNA molecules as described m Protocol 13. 99

3: RNA editing in mitochondria Prot ocol 13 . Assay of RNA ligase in mitochondrial extracts

Reagents e Mitochondrial extract see Protocol 11 I

m . TS, or S-1 00;

3000Ci.m tmol, 10mCi mil

e Cytosolic RNA substrate 1500 ~Jglml ) prepared as described in Protocol 12 • 10mM ATP

• 1 .OM MgCI2 e 0 . 25 M Hepes- KOH, pH 7 .9 • 0 . 1 M OTT

• ( a · 32P I pCp (New England Nuclear;

e OMSO • RNAguard IPharmacia, # 27-0815·0 1 331Ji ml) • DE81 discs and 0 .5 M sodium phosphate buffer, pH 6.8, wtth 0 . 5% sodtum pyro phosphate (see Protocol 9 1

Method 1. In a microcentrifuge tube on ice, m1x: • cytosolic RNA substrate

1-51JI

e 1 .0 M MgCI 2 e 0 . 1 M OTT

2 Ill 3.31JI

e 0 .25 M Hepes-KOH, pH 7.9

20 Ill

• lOmMATP e OMSO

11JI 10111

• [a- 32P]pCp e RNAguard • mitochondrial extract • H20

2111 1 IJI 1 0 IJI to 50 Ill final volume

2. Incubate the mixture at 4 °C for 1 5 h. 3 . Measure the addition of [a-32P]pCp to the RNA substrate by spotting aliquots of the mixture on DE81 filter discs and processing them as described in Protocol 9 , steps 4-6.

8.5 Cryptic RNase A sequence- or structure-specific cryptic RNase activity can be detected in mitochondrial extracts (TL, TS, or S-1 00 extracts, see Protocol 11 ). This en ptic RNase can be activated either by the addition of heparin (5 )Jg/ ml) o by predigestion of the lysate with proteinase K. Protoco/14 describes the activation of the cryptic RNase and its subseq tent assay. The RNA substrate used for this assay is a 200 nt RNA synthesized b> in vitro transcription from a n bacteriophage promoter using T7 RNA polymerase. The template DNA consists of the 22 nt PER of the cytochrome b gene together with 56 nt of 5 ' flanking sequence, 186 nt of 3 ' flank ing sequence, and 73 nt of Bluescript vector sequence at the 5' end. Cleavage bY the cryptic RNase occurs at one major site and four minor sites within the P ER· 100

Larry Simpson. Agda M. Simpson, and Beat Blum Protocol 14. Assay for a sequence- or structure -specific cryptic RNase activity in mitochondrial extracts

Equipment and reagents Smal site of pBiuescript SK l- l vector from Stratagene)

• Mitochondnal extract (Tl, TS, or S- 100, see Protocol 77 1 • Hepann (S1gmal • Proteinase K tBRL): dissolve the prote1nase K at 10 mgfml in 50 mM Tns-HCI, pH 8.0. 1 mm CaCI 2 e 10 mM ATP e 10 x buffer {30 mM MgCI2, 0.1 M TrisHCI, pH 7. 5, 50 )Jgfml heparin) • pNB2 plasmid [a 198 bp Acci!Rsal restriction fragment from the plt120 maxicircle region containing the 5 '-end of cytochrome b gene cloned into the

e pNB2 RNA substrate (108 c.p.m 11Jgl: transcribe BamHI·digested pNB2 plasm1d DNA in vitro us1ng T7 RNA polymerase and (a- 32P]UTP as described 1n Volume I, Chapter 1 !Protocol4 ) or Chapter 2, thiS volume (Protocol 4 I • Phenol:chloroform ( 1: 1, vlvl and 2M NaCI as Protocol 3 • 8% polyacrylamide/7 M urea denaturing (sequencing) gel, loading and electrophoresis buffers and equipment (see Volume I, Chapter 1, Protocol 8 or Volume I. Chapter 4, Protocol 11 I

Method Activation of the cryptic RNase 1. Either add proteinase K (1 00 l)g/ml final concentration) to the mitochondrial extract and incubate the extract for 5 min at 37 °C or add heparin to 5 ~Jg /ml. Activation by protease predigestion and heparin are synergistic and together result in better cleavage.

2. In a microcentrifuge tube on ice, mix: 5 ~I 51)1

• 10 x buHer e lOmM ATP pNB2 RNA substrate ( 10 4 c.p.m.)

1 ~JI

• activated mitochondrial extract (from step 1)

10 ~I

• 32P-Iabelled

to 50~~ final volume

• H2 0 3. Incubate the mixture for 1 h at 27 °C.

4. Phenol extract the RNA and recover the RNA by ethanol precipitation as described in Protocol 3, steps 13- 16. 5 . Add gel loading buffer ( 10 IJI of 10M ureal and analyse the cleavage products by gel electrophoresis and autoradiography as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 11.

8.6 gRNA:m.RNA chimaera-forming activity In order to study the first step of the proposed trans-esterification model for RNA editing (16, 17), the authors developed a cell-free system using an extract from mitochondria prepared by sonication followed by salt extraction. Chimaera formation is monitored by the covalent transfer of uniformly-labelled 32P-gRNA 101

3: RNA editing in mitochondria to a higher molecular weight non-radioactive test mRNA by gel electrophoresis. Both the 32 P-gRNA and the test mRNA are synthesized by T~ transcription from PCR-derived DNA templates (63). Sequence analysis of the reaction products by selective PCR amplification and cloning revealed gRNA:mRNA chimaeric molecules as the most prominent products (20). This in vitro system has been used to confirm the importance of the anchor sequence between the gRNA and mRNA, and should be useful for developing conditions for complete in vitro editing (20). The system is described in Protocol 15.

Protocol 15. gRNA:mRNA chimaera-forming activity

Equipment and reagents • Centricon 10 centrifugal centrators (Amicon 4205)

microcon-

• Kinetoplast-mitochondrion fraction isolated from one litre of L. tarentolae according to Protocol 6. Keep the kinetoplast-mitochondrion fraction at - 70 °C until extraction is performed 32

•Uniformly-labelled P-gRNA prepared by in vitro transcription with [ a-32P) UTP and T7 RNA polymerase (Volume I, Chapter 1, Protoco/4 or Chapter 2, this volume, Protocol 4) and isolated by gel electrophoresis (Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4,

Protocol 1 1) • Test mRNA containing the PER plus flanking (anchor sequence) region; prepare this by in vitro transcription with T7 RNA polymerase followed by gel electrophoresis (see above) e1.0 M Hepes-NaOH, pH 7.9 e 0. 5 M EDTA (adjust the pH to 8.0 w 1th NaOH)

e Q.1 M DTT e 0 .1 M PMSF (Sigma # P-7626) in iso· propanol • Human placental RNase Inhibitor IBRL #5518SA) • 20 mg/ml proteinase K (BRL # 5530UA ) stock • Hypotonic buffer (1 0 mM Hepes-NaOH, pH 7.9, 0.5 mM EDTA) • Extract buffer (20 mM Hepes-NaOH. pH 7.9, 20% glycerol, 0.1 M KCI, 0.2 mM EDTA, 0.5mM PMSF, 0 .5mM DTT prepared fresh from stock solutions) • 2 x salt extraction buffer (40 mM Hepes, pH 7.9, 50% glycerol. 0.84 M NaCI. 0.4 mM EDTA, 1 mM PMSF, 1 mM DTT. prepared fresh from stock solutions) • Annealing buffer (20 mM Hepes-NaOH. pH 7 .9 , 0 . 1 M KCI, 1 mM EDTA) • Reaction buffer (8% PEG 8000, 12.5 mM MgCI 2 , 2. 5 mM A TP. 1 unitl~l RNase inhibitor) • Proteinase K solution (0.25% N-lauryl sarcosine, 25 mM EDTA , 0.25 mglml proteinase K)

• Glycerol (ultrapure; BRL 5514UA) e2.0 M NaCI e1 .0 M KCI

• Sonicator (Braunsomc microtip; Braun)

e0. 1 M MgCI2 •13% (wl v) polyethylene glycol 8000 (PEG 8000; Sigma #P-2139) • N-laurylsarcosine (Sigma # L-51 25)

# 451 0

w ith

• Materials and equipment for poly acrylamidel urea gel electrophoresiS as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4 .

• 20 mM ATP (Pharmacia # 27-2056-01)

Protocol 11

Continued 102

Larry Simpson. Agda M. Simpson, and Beat Blum

Protocol 15. Continued Method 1 . Resuspend the kinetoplast-mitochondrion fraction from one litre of cell culture in 2 ml of 1ce-cold hypotonic buffer. Keep this on ice for 10 min to allow the mitochondna to swell. 2 . Disrupt the swollen mitochondria by sonication at 5 °C using three 20 sec periods at 100 watts. 3 . Immediately add 2 ml of 2 x salt extraction buffer and gently agitate the solution with a small magnetic stirrer for 30 min on ice.

4. Clarify the extract by centrifuging at 50 000 g for 30 min at 4 °C. 5 . Concentrate the extract at 4 °C on two Centricon 10 microconcentrators to an approximate vel. of 100 IJI each.

6 . Change the buffer at 4 °C using the same microconcentrators by applying two 2 ml lots of extract buffer to each. 7. Quick freeze 200 IJI aliquots of the pooled extracts in dry ice/ethanol and store them at - 70 °C. Chimaera-forming activity is maintained for up to 3 months. 32P-Iabelled gRNA and the test mANA in 2.51JI of annealing buffer. Denature the RNA for 3 min at 70 °C then anneal them at 37 °C and 25 °C for 10 min each.

8 . Anneal equimolar amounts of the

9. Add 81JI of reaction buffer and 1 5 IJI of thawed mitochondrial extract {from step 7) to the annealed RNAs. Incubate for 15-120 min at 27 °C.

10. Stop the reaction with 100 IJI of proteinase K solution and incubate at 37 °C for 20 min.

11 . Recover the RNA by phenol extraction and ethanol precipitation as described in Protocol 3, steps 13- 16.

12. Analyse the resulting RNA chimaeric products on denaturing acrylamide/ urea gels followed by autoradiography {Volume I. Chapter 1, Protocol 8, or Volume I, Chapter 4 , Protocol 11 ).

The RNA products from Protocol 15 may also be analysed by sequence determination following RT-PCR and cloning. In order to amplify specifically the exogenous RNA, a 'tag' sequence may be added to the test RNA. Further details of RT -PCR can be found in Volume I, Chapter 2, Section 3.4 and Volume I, Chapter 3, Section 2.2.3

Acknowledgement This work was supported in part by NIH research grant Al09102.

References I. Hodges, P. E., Navaratnam, N., Greeve, J. C. and Scott, J . (1991). Nucleic Acids Res., 19, 1197. 103

3: RNA t>diting in mitochondria 2. Lau, P. P, Xiong.\\ . Zhu, H.-J ., Chen, S -H .• and Chan, L. (1991) J. B IJ/, Chem., 266, 20 550. 3. Gualberto, J. M., Lamattina, L., Bonnard, G., Wei!, J . H., and Greinenberger, J M. (1989). 1\'ature, 341, 660. 4. Covello, P . S. and Gray, M. W . (1989). 'Vuwre. 341, 662. 5. 'vtahendran, R., Spothwood, '.1. R, and \Iiller. D L. (1991) \'ature, 349, 43.;. 6. Simpson L. and Shav., J. (1989). Cell, 57, 355 7. Vidal, S., Curran, J., and Kolakofsky, D . (1990). EMBO J. , 9. 2017. 8. Driscoll, D., Wynne, J ., Wallis, S., and Scou, J. (1989). Cell, 58, 519. 9. Chen, S.-l·l., Li, X, Liao, \\' . S. L., Wu, J . H .. and Chan L. (1990) J. Btol. Chem .. 265, 6811 10. Lau, P. P., Chen, S.-H., Wang, J. C., and Chan, L. (1990). Nucl. Acids Res 18, 5817. II. Smith. H. C .. Kuo, S.-R .. Backus. J. W . Harris, S. G., Spark , C. E., and Sparks, J D. (1991) Proc. /'vat/ Acad. Sci. USA, 88, 1489. 12. Greeve, J., Navaratnam, .. and Scott, J. (1991) . •Vue/, Acids Res. , 19, 3569 13. Backus, J. W. and Smith, H . C. (1991). Vue/. Acids Res.. 19, 6781. 14. Simpson, L. (1990). Science, 250, 512. 15. Blum, B., Bakalara, N., and Simpson, L. (1990} Cell, 60, 189. 16. Blum, B, Sturm,, . R., Simpson, A. \1 .. and Simpson L (1991). Cell. 65 3. 1'. Cech, T R. (1991). Cell, 64, 667. 18. Koslowsky, D J, Gringer, H . U., Morales, T. H., and Stuart, K. (1992). Nature. 356, 807 19. Harris, M. E. and HaJduk, S. L. (1992}. Cell, 68, 1091 .:!0. Blum. B. and Simpson, L. (1992). Proc. \'all A cad. Set L.SA. 89. II 9~ 21. Simpson, L (1987). Annu. Rei'. .\1tcrobtol.• 41, 363. 22. Sturm, N. R. and Simpson, L. (1991). Nucl. Actds Res., 19, 6277. 23. Pollard, V. W., Rohrer, S. P., Michelotti, E. F., Hancock, K., and Hajduk, S L. {1990). Cell, 63, 783. 24. Kidane. G, Hughes, D, and Simp~on, L {1984) Gene, 27. 265. 25. Maslo", D. A. and Simpson, L. (1992). Cell, 10, 459 26. Morel, C., Chiari, E., Camargo, E., Mattei, D., Romanha, A . and Simpson, L. (1980). Proc. Nat/ Acad. Set. USA, 77, 6810. 27. Sturm. "1. R. , Degra\e, W., Morel, C .. and Simpson, L (1989). Mol Bwc rem. Parasitol., 33. 205. 28. Gomez-Eichelmann. M. C., Holz, G., Jr , Beach, D., Simpson. A. M .. and Simpson, L. (1988). Mol. Biochem. Parasitol., 27, 143. 29. Simpson, L. (1972). Int. Rev. Cytol., 32, 139. 30. Stuart, h. 0991). A111111. Re1·. .\llcrobiol. 45 327 31. Benne, R (1989). Biochim. Bwphys. Acta, 1007, 131. 32. Feagin, J and Stuart. K. (1988). ~to/. Cell Bioi., 8, 1.259 33. Feagin, J., Jasmer, D., and Stuart, K. (1987). Cell, 49, 337 34. Duszenko, M., Ferguson, M ., Lamont, G., Rifkm. M., and Cros'i. G. (1985). J. £r:p. \1ed., 162, 1256. 35. Briones, \.1. R. S., Nelson, K., Beverle}. S. M , .\ffonso. H. T. Camargo. E. P .. and Floeter-Winter, L. M. (1992}. 'viol. Biochem. Parosilol., 53, 121 . 36. Simpson, L. and Berliner, J . (1974). J. Protozoal., 21, 382. 37. Simpson, L. and Simpson, A (1974) J Proto::.ool., 21. 7i'4.

104

Lorry Simpson, Agdo M. Simpson, and Beat Blum 38. Simpson, L (1979). Proc '\'at/ Acad. Sci. USA, 16, 1585. 39. Ryan, K A., Shapiro, T. A., Rauch, C. A., and Englund, P T (1988). Annu. Re~ . Microbtol., 42, 339. 40. Hajduk, S., Klein, V., and Englund, P. {1984). Cell, 36, 483. 41. Tibayrenc, M., \\ard, P ., Moya, A ., and Ayala, F. (1986). Proc . .Vat/ Acad Set. USA, 83, 115 . 42. Brunk, C. and Simpson, L. (1977). Anal. Biochem. , 82, 455 43. Goncalves, A . M., Nehme, N. S., and Morel, C. M. (1984). In Genes and antigens of paraslles (ed. C. M. Morel), p. 95. Fundacao Oswaldo Cruz, Rio de Janeiro. 44. Beidler, J. L., Hilhard, P. R., and Rill, R. L. (1982). Anal. Biochem, 126, 374 ~5. Lopes, U., Momen, H., GrimaldJ, G., 1\larzocht, M., Pacheco, R , and Morel, C. (1984). J. Parasitol., 70, 89. 46. Morel, C. and Simpson, L. {1980). Am. J. Trop. Med. Hyg., 29, 1070. 47. Avila, H., Goncalves, A.M., Nehme, N. S., Morel, C. M ., and Simpson, L. (1990). Mol. Biochem. Parasito/., 42, 175. 48. Simpson, L. {1968). J. Proto:.oo/., 15, 132. 49. Braly, P ., Simpson, L., and Kretzer, F (1974) J. Proro;:ool, 21, 782. 50. Bakalara, N., Simpson, A.M., and Simpson, L. (1989). J. BIOI. Chem., 264, 18 679. 51. Simpson, L. and Simpson. A. (1978). Cell, 14, 169. 52. Simpson, A. \1., Suyama, Y , De\~e~. H., Campbell, D. and Simpson, L. (1989) Nucl. rlctds Res. , 17, 542.,. 53. Hancock, K. and Hajduk, S. L. (1990). J. Bioi. Chem., 265, 19 208. 54. Maslov, D. A., Sturm, N. R. • Niner, B. M., Gruszynski, E. S., Peris, M., and Simpson, L. ( 1992). ~1o/. Cell. Bioi., 12. 56. 55. Sturm, . R. and Simpson, L. (1990). Cell, 61, 8"1. 56. Sha\\ , J , Campbell, D., and Simpson, L. (1989). Proc. Sat Acad. Set., 86,6220. 57. Blum , B. and Simpson, L. (1990). Cell. 62, 391. 58. Masuda, H .. Simpson, L., Rosenblatt. H., and Simp~on, /\. (1979). Gene, 6, 51. 59. \\ood, W, Gitschier, J ., La5k)', L., and La\""· R. (1985). Proc. Nat/ Acad Sci USA, 82, 1585. 60. Abraham, J., Feagin, J., and Stuart, K. (1988). Cell, 55, 267. 61. Decker, C. J. and Sollner-Webb, B. (1990). Cell, 61, 1001. 62. McPherson, M. J., Qutrke, P., and Taylor. G. R. (ed) !1991). PCR: a practical approach. IRL Press, Oxford. 63. 1\.·f llljgan, 1. F., Groebe, D. R., Witherell. G. W., and Uhlenbecl.:. 0. C. (1987). Nuc/. Actds Res., IS, 8783. 64. Von Haeseler, A .. Blum, B , Simpson, L .. Sturm,'-· and \\aterman. M.S. (1992). 'Yuc/. Actds Res.. 20, 2717. 65. Sha\\. J , Feagm, J . E., Stuart, K .. and Simpson, L. (1988) Cell, 53, ~01 66. \an der Spek. H , ArtS, G.-J ., ZwaaJ, R. R., Van den Burg, J . , Sloof, P ., and Benne, R. (1991). EMBO J., 10, 1217. 67. Simpson, A M , Bakalara, N .. and Simpson, L. (1992). J Btol Chem., 267,6782 68. KoslO\\Sky, D J., Bhat, G J .. Read, L. t.., and Stuart, K (1992). Cell, 61, 5r 69. Rc:ad, L. K., Myler, P. J ., and Stuart, K. (1992). 1. B10/. Chem, 267, 1123. 70. Read, L. K., Corell, R. A., and Stuart, K. (1992). Nuc/. Acids Res., 20, 2341. 71. White, T. and Bor~t. P. ( 1987). Nuc/. AC'ids Res. , IS, 3275.

105

Analysis of messe11ger RNA turnover in cell-free extracts from mammalian cells JEFF ROSS

1. Introduction Messenger RNA turnover innuences gene expression in most or all cells, from bacteria to mammals (I, 2). Two sorts of observations support this notio n . First, the steady-state levels of many mRNAs are determined more by their half-lives than by the rates at which their genes are transcribed. Second, the half-lives of some mRNAs are not fixed but vary in response to the nutritional status of the cell, its position in the replication cycle, or its stage of differentiation. Many important questions need to be answered to understand how mRNA turnover is regulated and how it innuences cell growth, differentiation, and neoplastic transformation. How many mRNA-degrading enzymes exist per cell? Which are endonucleases and which exonucleases, and at what site in a mRNA molecule do they first begin the degradation process? What is the pathway of mRNA decay, i.e. what are the degradation intermediates? What sequences in a mRNA determine its half-life? How does translation affect mRNA turnover? What trans-acting factors regulate mRNA turnover and how do they function? These questions illustrate the need to use in vitro systems to investigate mRNA turnover, particularly in cells of higher organisms in which genetic analysis is difficult. In ~·itro systems offer several advantages (Table 1). Two of these are particularly imponant. (a) mRNA decay intermediates that are difficult or impossible to detect in intact cells can sometimes be identified in cell-free systems. The average intracellular half-life of an mRNA might be 20 h, but, once each individual mRNA molecule begins to be destroyed, the n:action is apparently complete in minutes or seconds. If degradation occurrc!d sJo,\ly, it would be eas) to detect mRNA decay intermediates in cells, which, with few exceptions, is not the case. Since degradation rates are slower in vitro than in cells, it is possible to identify intermediates.

4 : mRNA turnover in cell-free extracts Table 1. Advantages of analysing mANA stability in cell -free extracts • mANA decay intermediates which are difficult or impossible to observe in cells can be detected in VItro. • Messenger ribonucleases and trans-acting factors which stabilize mANA can ,e assayed and purif1ed • Relat1ve mANA turnover rates can be measured Without us1ng mh1b1to ·s of transcnpt1on. most or all of which are toxic to cells and not specific for transcnption. • The effects of mANA sequences and structures on half-life can be readily mvest1gated.

(b) In vitro mRNA decay systems, Like in vitro transcription systems, permn the investigator to detect and purify enzymes, co-factors, and trans-acung regulators. Even if practical schemes are available to select mutations m genes involved in mRNA turnover, some of the interesting mutants m1~ht be lethal, making it necessary to devise in vitro assays to characterize the relevant gene products. This chapter focuses on three in vitro methods currently used to investigate mRNA turnover in cells of higher organisms. It is intended as a guide and by no means presents all possible assay schemes. Improvements on the curre!l! systems \Hll surely continue to be made. The advantages and disad' amages of each system wiU need to be considered by the investigator in the context of the experiments. ln choosing a suitable in vitro system, it is important to remem ber that relative mRNA half-lives in vitro should re nect those in cells: mR NAs that are stable in cells should also be stable in vitro and, conversely, unstable mRNAs should be unstable in both. Half-Hves in cells and in vitro are unlikely to be identical, since mRNA decay occurs more slowly in vitro. However, if LWO mRNAs are degraded at rates differing by 20-fold in cells but by 3-fold or le)s in vitro, then the extract or reaction conditions should be modified. For example, mRNA-specific regulatory factors might have been inactivated during extract preparation. The protocols in this chapter suggest some options for modifymg each system.

2. Choice of mRNA substrate The choice of substrate (endogenous or exogenous) depends on the experimeutal question being asked and on various parameters such as the number of available cells, the amount per cell of the mRNA of interest, and the activity of 'nonspecific' cellular RNases (Table 2). NB. The term non-specific RNase denotes an activity capable of degrading all RNAs (rRNA, tRNA, or mRNA) at sim1Jar rates in vitro. The term is not meant to imply lack of function for such an RNase. only that it is not yet understood what its function might be. 108

Jeff Ross Table 2 . Use of endogenous and exogenous substrates for in vitro mANA turnover stud1es Endogenous substrate (mA NP, either free or polysome-associated)

Adltantages • ' authentic' substrate, made by cellular transcription :modification enzymes e known to be respons1ve to at least some trans-acting regulatory factors in vitro

Oisi1dvantages • d ifficult to detect 1f the mANA is scarce • prone to degradation during isolation • cumbersome when variants need to be analysed Exogenous substrate (protein-free cellular mANA or m vitro synthesized RNA)

Adv·anrages • easy to detect, especially if radiolabelled • ectsy to prepare variants

Disadvantages • translated w ith low efficiency in some extracts • m ight not respond to regulatory factors

2.1 Endogenous substrates (free mRNP or polysome-

associated mRNP) Polysomes contain the substrate messenger ribonucleoprotein (mRNP) to be analysed and often the messenger RNases as weU. There are several advantages to using endogenous mRNP substrates. First, the nature of the substrate is relatively clear, i.e . the mRNA was synthesized by the celJ and is competent for translation, since it is polysome-associated. Therefore, within the constraints imposed on any subcellular fraction prepared in the laboratory, an endogenous subst rate could be considered more or less authentic. Second, some endogenous substrates respond in vitro to factors involved in regulating mRNA stability (3-7). ln contrast, the turnover of some exogenous, deproteinized mRNA substrates is unaffected by such regulatory factors (see Section 2.2). There are three potential disadvantages of endogenous substrates. First, if th•e mRNA is scarce, large amounts of polysomes might be required to deteC't it. Second, polysomes containing undegraded mRNA can be difficult to prepare, especially from some primary animal tissues, unless special techniques are devised to red uce non-specific RNase activity (8). Third, it might be difficult or impossible to engineer cells to express interesting modified substrates, for example, mRNA that has a discrete 3 ' terminus and that lacks a polr(A) tract. 109

4 : mRNA turnover in cell-free extracts

2.2 Exogenous substrates The assa} is relatively easy with an exogenous substrate, especially if it is radiolabelled. The substrate is simply incubated '' ith a cell extract, purified. and analysed by gel electrophoresis. Exogenous substrates can be modified more easil} than endogenous mRNAs, "ithout concern for the effects o; the modifications on transcription, capping, or transport to the cytoplasm For e\ample, substrates of almost any size and sequence can be generated rapidly by cutting a eDNA clone at different sites and transcribing each template '' ith an appropriate bacteriophage RNA polymerase. HO\\e\'er, there are two important disadvantages of exogenous substrates. First, mRNA turnover and translation are often linked (I, 9, 10), but it ts difficult to translate exogenous mRNA efficiently in extracts from nucleatea mammalian cells. Second, some exogenous substrates, unlike their endogenou~ counterparts, fail to respond to regulatory factors in 1•itro. For example. polysome-associated (endogenous) c-myc mRNP is regulated in vitro by cytosolic destabilizers (3, 7) but deproteinized (exogenous) c-myc mRNA is not (G. Brewer and J. Ross, unpublished observations).

3. Preparation of undegraded mRNA and polysomes When using an endogenous substrate, it is important that the mRNA and polysomes prepared are undegraded in order to obtain meaningful data. Contamination of glassware or reagents with RNases, which seem to be ubiquitous, can ruin a preparation . The fo llowing precautions, coupled with a large dose of common sense, will help maintain RNA integrity: (a) Choose the proper cell or tissue. Some cells contain more non-specific RNases than others, so even the most careful investigator might have difficulty isolating intact polysomes from them . In general, tissue culture cell lines contain less RNase acrivity than animal tissues. If necessary, various RNase inhtbitors are available. Two of them, placental RNase inhibitor and diethylpyrocarbonate (DEPC), are discussed in this chapter. Vanadyl ribonucleoside complex at 4 mM and heparin at 0.2 mg/ ml have also been exploited to block RNase activity during preparation of extracts from anmtal tissues (8). (b) Use sterile plasticware whene,er possible. (c) Wear disposable plastic gloves at all times, since bod} surfaces and tluids comam active RNase(s). (d) Pre-rinse all glassware with DEPC at least one day before use (see Protocol 1). DEPC treatment is recommended, because autoclaving migdt not inactivate all RNase contaminants in the glassware. Moreover, DEPC is an excellent sterilizing agent. 110

leff Ross (e) Filter all solutions and store them at 4 oc or at - 20 °C. MagnesiUm iom, and carbon sources (e.g. sucrose) might permit growth of certain microorganisms, all of which contain RNases. The best way to handle these solutions is to sterilize them by filtration through a micropore ( ~0.451Jm) membrane (e.g. Millipore HA\\ P) in a tissue culture hood. Store them m a refrigerator, only open the stock bottles in a sterile hood, and dispense them using Sterile pipettes. Tape a note on each bottle stating that the solution is sterile and must be opened only in a hood. Some laboratories pretreat their glass-distilled water by adding DEPC to a final concentration of 0.02~o and then autocla,·ing. HPLC-grade ''ater or \\ater prepared by deionization and double distillation might be suitable for use without sterilization or DEPC treatment. (f) Carry out all procedures at 2-4 °C, from the time the cells are hanested until the extracts are placed in the - 70 oc freezer.

Protocol I describes the treatment of glassware '' ith DEPC and b> baking. Protocol 1 . Treatment of glassware to inactivate RNases.

1. Add a small volume of DEPC to each 1tem of glassware. 2 . Rotate the glassware so that all inner surfaces are exposed to the DEPC. 3 . Caution . DEPC can form a carcinogenic compound by reacting w1th primary amines. Therefore, after rinsing the glassware, pour the DEPC into a beaker

in a hood, add excess water, and then let the liquid evaporate to dryness. 4 . Rinse the glassware thoroughly with distilled water until all traces of the distinctive DEPC odour have d1sappeared (a min1mum of ten rinses should do). 5 . Bake the glassware in a 220 °C oven for at least 1 h, preferably overnight.

4. Preparation of cell extracts for in vitro mRNA

decay reactions There is no single, universally accepted standard method for analysing mRNA turnover in vitro, nor is one likel} to evolve. Different e>."tracts and reaction conditions are required to account for variables which can influence mRNA turnover within the cell. These variables, in turn, depend on the hormonal and nutritional status of the ceil, its position m the cell cycle, and its stage of differentiation. For example, the half-lives of some hepatic cell mRNAs can fluctuate by 30-fold or more when certain hormones are added to or removed from the culture medium. The following questions are relevant to the stability of most mRNAs and must be considered in deciding how best to carry out cell-free mRNA decay reactions. 111

4:

e Is

mRNA turno,·er in cell-free extracts

the mRNA translated in cells and, if so, at what rate?

• Does the cell contain trans-acting regulatory factors which inOuence the mRNA half-life? If so, are these factors freely diffusible throughout the cytosol or are they associated with particular cytoplasmic components such as ribosomes, endoplasmic reticulum, the mRNA itself, etc.?

e Is the mRNA itself localized as a result of its association with microfilaments. endoplasmic reticulum, etc.? Three methods are described for preparing extracts. Each has advamagt and disadvantages, depending on the mRNA to be analysed and the variable' cited above. The investigator must choose the appropriate extract or combination of extracts, substrates, and in vitro conditions so as to reOect as closeh as possible the circumstances under which the mRNA is degraded withtn the cell. The first method exploits ultracentrifugation to prepare polysomes and post-polysomal supernatant (Sl30) from cells homogenized in a detergent-free, low-salt buffer (see Protocol 2). A major advantage of this method is us nexibility. Since polysomes contain mRNA and messenger R 'ase(s), t '1e turnO\er of polysome-associated mRt As can be analysed simpl}' by incubati 1g polysomes under appropriate conditions (see Protocol 6). The S 130 seems to ha\e little messenger RNase activity but does contain soluble, trans-acting factors which regulate the half-lives of certain mRNAs. These factors can be assayed in reactions containing mixtures of SJ30 and polysomes (see Protocol 6 . Exogenously added, radiolabelled mRNA can also be assayed in reaction~ containing polysomes or polysomes plus Sl30 (see Protocol 7). Ribosome-bound messenger RNases can be separated from polysomes by exposing the polysomes to high salt, pelleting them in an ultracentrifuge, and harvesting the supernatant (see Protocol 3). The high-salt extract can then be used to analyse the decay of radiolabelled mR A (see Protocol B). One disadvantage of using detergentfree polysomes is a very lo\\ translation rate, both for endogenous and exogenou' mRNAs. The second method for preparing extracts uses lysolecithin to permeabilize adherent ussue cullure cells, which are then scraped from the culture di~h. broken, and brieO) centrifuged to remove nuclei (see Protocol4). These extra ts are easy to prepare, and they support translation of endogenous and exogeno ts mRNAs more efficiently than detergent-free extracts (see ProTocols 9 and 10 ). One disadvantage is a lack of information on whether lysolecithin extracts retain their translational and mRNA turnover activities \\hen they are fractionated or frozen. The third method u~es a lysate prepared from reticuloc)1e , \\hich arc immature. enucleated red blood cells contairung abundant polysomes (see Protocol 11 ). This system is the most active of the three for translation, and reticulocyte extracts can be purchased at modest cost. The lysolecithin and reticulocyte extracts can be made mRNA-dependent by exposing them brieOy 112

]eft Ross to micrococcal nuclease, which eliminates the mRNA but not the ribosomes or other components necessary for translation (see Protoco/5). After inactivating the micrococcal nuclease, the extract can be incubated with exogenous mRNA under conditions permitting active translation and the half-life of mRNA which had become polysome-associated can be determined (see Protocols 10 and 11). Micrococcal nuclease treatment of detergent-free extracts is fruitless, because translation initiation occurs so inefficiently. It is important to stress once again that these methods should be used as guides and may need to be modified to suit specific experimental parameters. Flexibility is essential and extracts can, in theor}, be mixed in various combinations. For example, if an investigator wishes to analyse the turnover of a mRNA which is being actively translated, radiolabelled mRNA can be incubated in nuclease-treated reticulocyte extract. However, suppose that the investigator suspects that the intracellular half-life of this mRNA is influenced by a cytosolic trans-acting regulatory factor, which is likely to be presem in S 130. Since reticulocytes are end-stage cells whose S 130 contains primarily haemoglobin, they are unlikely to have retained all of the S 130 regulatory factors which are present in undifferentiated, nucleated cells. Therefore, the best approach might be to mix the mRNA to be tested with reticulocyte extract plus S 130 isolated by detergent-free lysis of nucleated cells. The multicomponent reaction mix might reflect the intracellular conditions to which the mRNA is normally e'posed (efficient translation plus a soluble regulatory factor) more accurately than reactions containing only detergent-free polysomes (minimal translation, no soluble factors) or detergent-free polysomes plus S 130 (soluble factors present, but minimal translation). Protocol 2 and the following protocols pertain to tissue culture cells, since limited information is available on the use of extracts from animal tissues for cell-free mRNA decay. In fact. it is unclear whether these protocols can be used with primary animal tissues. Investigators wishing to use animal tissues should take special care to reduce non-specific RNase activity. The following RNase inhibitors might be useful: • Placental or liver RNase inhibitor (II), available from Amersham, Boehringer Mannheim, Calbiochem, ICN Biomedicals, Promega, Stratagene. Useful amounts must be determined empirically, but an appropriate starting point is 100 units per mi. • Ribonucleoside-vanadyl complexes (12), available from Bethesda Research Laboratories, Ne\\ England Biolabs, Sigma. Starting point: 2.5 roM. Moreover, buffers can be designed to contain combinations of inhibitors which might be more effective than a single inhibitor. The composition of one buffer used to isolate intact polysomes from primary cultures of amphibian liver is given below (8): 113

4: mRNA turnover in cell-free extracts • 0.2 M sucrose e 70m~1 KCI e IOmM MgCI2 e SmM NaCl • 4 mM vanadyl-ribonucleoside complex e l mM EGTA • 0.2 mg/ml heparin el mM DTT • 100 units/ml placental RNase inhibitor e SO mM Hepes-NaOH, pH 7.8

Protocol 2 . Preparation of polysomes and post-polysomal supernatant (S 1 30) from exponentially-growing tissue culture cells lysed without detergent8

Equipment and reagents NB All buffers and tubes should be at 2-4 °C prior to the experiment pestle with a clearance of approximately 0.1 mm; use a vessel with a capacity 2to 3-fold greater than the volume of Solution A used for cell lysis (see step 6 1; a Dounce-type glass homogenizer with a tight-fitting pestle (clearance 0.06 mml may also be required (see step 17)

• Exponentially-growing tissue culture cells • Solution A (1 mM potassoum acetate, 2 mM magnesium acetate, 2 mM DTT, 10 mM Tris-acetate, pH 7.6 at room temperature) • Solution 8 (solution A containong 30% (w/v) RNase-free sucrose) • Tissue culture medium without serum or antibiotics; medium Ham's F 12 (Flow, Gibco, HyCione) has been used in the author's laboratory, but any osotonic growth medoum should do • Potter-Elvehjem homogenozer with Teflon

• Low-speed centrifuge, ultracentrifuge, and appropnate tubes; polyallomer ultra centrifuge tubes are preferable; they need not be baked but should be thoroughly rinsed with RNase-free water and then drained dry immediately pnor to use

Method 1. Count the cells. 2. Centrifuge the culture for 4 min at 34 g (average I. corresponding to 500 r.p.m 1n a low-speed centrifuge with a swinging bucket radius of 8 em. Note: This and all subsequent procedures should be performed at 1-2 ° C. All buffers should be kept ice cold throughout the manipulations. 3. Asporate and discard the supernatant. 4. Gently resuspend the cells in ice-cold tissue culture meoium without serum or antibiotics. Centrifuge as in step 2. 5. Repeat steps 3 and 4.

Continued 114

Jeff Ross Protocol 2 . Continued 6 . Resuspend the cHIIs in solution A. The volume will depend primanly on the cell number and the cell type, since some cells do not lyse easily by mechanical homogenization, especially if they are too concentrated (see step 8 below) . The maximum concentration of cells for high· efficiency homo~Jenization (95% or greater lysis) must be determined in a pilot experiment by counting the percentage of broken cells using a haemocytomet er and microscope. In most cases, 10 8 cells/ml is a reasonable starting point, but the optimum number might be up to 10-fold lower. 7. Transfer the cells to the prechilled Potter-Eivehjem homogenizer.

8 . Place the homogenizer vessel in an ice-water bath and break the cells by moving the pe!stle rapidly up and down and twisting it left and right at the same timc3. The number of up and down strokes required for efficient cell lysis will vary, depending upon the cell type and their concentration. As a starting point, 20 strokes should do (see step 6 above). Avoid generating excess bubbles. 9 . Centrifuge the homogenate for 10 min at 8300 g (average ), which corresponds to 9000 r.p.m. in a swinging bucket rotor, radius 9.1 em. 10. Remove and save the supernatant (S 10), taking care to avoid the pelleted material (nuclei and membranes). It is best to leave behind a small amount of supernatant so that none of the pelleted material contaminates the S 10. 11 . Gently layer the S 1 0 over a cushion of solution B in an ultracentrifuge tube. The volume of solution B should be approximately 25% of the tube capacity. If the tube is insufficiently filled with S 10, add solution A.

12. Centrifuge at 130 000 g (average). 2 °C, 2. 5 h, which corresponds to 36 000 r.p.m. in a Beckman SW60 rotor. If you intend to save the S 130, allow the rotor to decelerate without the brake. 13. Carefully remove! the supernatant (S 130) above the sucrose cushion, taking care not 1ro harvest the whitish material located on top of the cushion. Leave a small amount of S 130 above the cushion, if necessary. Do not be concerned if some of the lipid at the top of the tube is harvested along with the S 130. Set aside the S 130 in an ice bucket. 14. Aspirate the remaining liquid, including the sucrose, and discard it. A bluish-white pellet of polysomes should be visible in the centre of the tube bottom. 15. Lay the centrifuge tube on its side and cut it across with a razor blade approximately two-thirds from the top. Discard the top piece.

16. Gently add approximately 1 ml of solution A down the sides of the bottom part of the centrifuge tube. Gently move the tube back and forth to wash any contaminating matenal, including sucrose, from the upper surface of the polysome pellet. Aspirate or pour off the liquid. Repeat this washing step once. It is not necessary to perform these washing steps quickly, since the polysomes will not dissolve in the buffer, but it is necessary to add the buffer slowly and gently, so as not to dislodge the polysome pellet from the bottom of the tube.

Continued 115

4: mRNA turnover in cell-free extracts Protocol 2. Continued 1 7 . Resuspend the polysomes in a small volume of solution A . The volume will depend on the quantity of polysomes recovered and the subsequent experiments. As a rule. use a volume such that the polysomes from 10 6 cells will be resuspended per microlitre. Polysomes can be difficult to resuspend. They tend to be 'sticky', forming clumps that are difficult to disperse. First, try resuspending them by pipetting the liquid up and down, using an automatic pipettor with a 200 1-11 or 1 ml disposable tip. If visible clumps remain, transfer the material to a tight-fitting Douncetype glass homogenizer with a clearance of 0.06 mm. Several up and down strokes with this homogenizer should break up the clumps. 1 8 . Measure the optical density of the polysomes and S 130 at 260 nm and 280 nm and store both preparations in multiple aliquots at -70 °C. As a guide, polysomes isolated from a logarithmically growing human erythroleukaemia cell line (K 562) resuspended at 10 6 cells' polysomes per microlitre give an absorbance reading of 220-260 through a 1 em light path at 260 nm.

19 . To assess polysome integrity, RNA from a small amount of the preparation can be extracted and analysed by agarose gel electrophoresis (13. 14). The rRNA can be visualized by ethidium bromide staining ( 13, 14). The rRNA is intact if the 28S and 18S rRNA bands are discrete and if the band intensity appears to be approximately 2:1 (28S: 18Sl. • from refs 13, 14.

Protocol 3 . Preparation of ribosom al salt wash (RSW) from det ergent-free polysomes8

Equipment and reagents • Polysomes isolated as in Protocol 2, steps 1-16

• 4 .0 M potassium acetate

• Solutions A and B. and ultracentrifuge equipment as in Protocol 2

Method 1. Resuspend the polysomes in solution A. The polysomes need not be fully dispersed, as described in step 17 of Protocol 2, but they should be more concentrated. A reasonable starting point is 2-5 x 109 cells' polysomes per millilitre. but higher concentrations can be used, if desired.

2. Transfer the polysomes to a small test tube or beaker containing a stirring bar, in the coldroom. Prior to use, the stirring bar should be soaked for 3-5 min in DEPC and rinsed thoroughly with water. 3. Add sufficient 4.0 M potassium acetate dropwise and with gentle sttrring to bring the final concentration to 0.5 M.

Continued 116

jeff Ross Protocol 3 . Continued 4 . Stir gently for an additional 1 5 min in the cold. Avoid bubbles.

5 . Layer the material over an appropriate volume of solution B in an ultracentrifuge tube and centrifuge for 2. 5 h at 130 000 g (average). 2 ° C. 6 . Carefully harvest the RSW located above the sucrose cushion. Retain the salt-washed polysome pellet (see step 8). 7 . M ix it gently but thoroughly, determine the protein concentration b ( 15), and store the RSW in small aliquots at - 70 °C. 8 . If desired, the salt-washed polysomes in the pellet at the bottom of the tube (step 6) can also be harvested, as per Protocol 2 , steps 16-19. ' From ref. 14. b Kits available from Bio-Rad and Pierce contain reagents for the Bradford protein determination assay.

Protocol 4 . Preparation of crude lysate by homogenization with lysolecithin8

Reagents • Non-confluent. adherent tissue culture cells in 100 mm diam. tissue culture dishes; suspension cells can perhaps be used by substituting low-speed centrifugation and gentle resuspension for aspiration (see step 4 below) • Solution C (0. 15M sucrose, 33 mM NH 4 Cl , 7 mM KCI, 4.5 mM magnesium acetate, 30 mM Hepes- KOH , pH 7.4) • Solution D ( 100-300 IJg/mllysolecJthin (L-a-lysophosphatidylcholine, palmitoyl) in solution C )0

• solution E (0.2 M NH 4 CI, 20 mM magnesium acetate. 7 mM KCI, 1 mM OTT, 1 mM A TP (dipotassium salt), 1 mM GTP (sodium salt). 40 11M each of the common 20 amino acids, 0.1 mM 5 -adenosylmethionine, 1 mM spermidine, 10 mM creatine phosphate (dipotassium salt ), 40 units per ml of creatine kinase, 100 units of placental RNase inhibitor, 0 .1 M Hepes-KOH. pH 7.4)•

Method 1. Aspirate the medium from each dish of cells.

2 . Place the dish on ice and quickly wash the cells twice with 2- 3 ml icecold solution C, removing the liquid by aspiration. 3 . Add 1- 2 ml of solution D to the dish and leave for 60 sec on ice. Be sure that the entire surface of the dish is covered w ith liquid. 4 . Aspirate the solution, and gently invert the dish for 60 sec to drain the remaining liquid to one corner.

5 . Aspirate off the remaining solution D. 6 . Put the dish back on ice and add 200111 of ice-cold solution E. 7 . Scrape the cells from the dish with a standard rubber-tipped cell scraper and transfer the cell suspension to a microcentrifuge tube.

Continued 117

4 mRNA turnover in cell-free extracts Protocol

4 . Contmued

8 . Disrupt the cells by passtng them 1 0 times through a 2 5-gauge needle attached to an appropnately-sized hypodermtc syringe. Monttor cell lysis by mtcroscopy. at least lor the first few ttmes the extraction is performed. If asptratton mrough a needle appears to be too harsh (i.e if the nuclei are disrupted cells can also be lysed by aspirattng them repeatedly with a Pasteur ptpette.

9 . Centnfuge the cell lysate tn a mtcrocentnfuge in the coldroom for 1 min to pellet the nuclei.

10. Remove the supernatant. taktng care not to contamtnate tt Wtth pelleted material.

1 1 . Store the supernatant on ice in preparation for m VItro mANA decay reacttons. It may be possible to store the lysate in aliquots at - 70 °C but the effects of freezing and thawing on the translational or mANA turnover activity of these extracts have not been reported. Therefore, preliminary studtes mtght be required to determtne whether freeztng ts feasible. • From ref. 5. • The concentration of lysolecithm to be used depends on the type of cells and must be determtned emp,ncally, since excess lysolectthtn will reduce translattonal activity. while msufftcient l¥s01ectthin wtll not permeabilize the cells. Brown et a/. ( 161 used 100 ~g :ml for PK ce !s, a pig kidney hne, and 250 IJg/ml for BHK cells, a baby hamster kidney line. Krikonan and Read (51 used 300 ~g/ml for Hela cells. Try different concentrations and compare the capacity of each extract to translate endogenous mANA, using the method descnbed 1n Section 5.2 < The opttmum concentrations of magnesium, potassium. and ammonium tons for translation or mANA turnover seem to vary considerably depending on the source of the cell extract and so should be determined for each cell line (dtscussed in ref. 161. The concentrations described here have been opttmized for Hela cell extracts (51. Opttmum concentrat
Protocol

5.

Treatment of lysolecithin extract with micrococcal nuclease8

Reagents • Crude lysolectthtn extract !prepared as in Protocol 41 • Micrococcal nuclease '1 mg ml'" disnlled waterl: store at -20 °C; the spectfic activity should be at ·east 6000 untts per mtlhgram of prote.o

e 01MCaCI2

• 0 1 M EGTA dtssolved
Method 1 . To a reaction tube tn tee add the following: • lvsolectthin exuact 0 .98 ml e Q,l M CaCI,

l01JI

• micrococca' nuclease

10 Ill

Continued 118

left Ross Protocol 5 . Continued 2

M1x the tube contents thoroughly and transfer the tube to a 20 °C water bath.

3

Incubate for 1 5 min.

4. Add 21 111 of 0. 1 M EGTA and 10 111 of calf liver tRNA and m1x thoroughly. 5 . This extract can now be used for in virro mRNA turnover reactions . It might be possible to freeze the extract 1n liqu1d nitrogen, but preliminary experiments should be performed to determine the effect of freezing and thaw1ng on translation and mRNA turnover. • From ref. 5 .

5. Methods for performing m vitro mRNA decay reactions 5.1 Detergent-free extracts Three variables, the source of messenger R Nase activit}, the combination of subcellular fracuons, and possible modifications of the basic system, need to be considered. (a) Reaction mixtures must contain either crude post-nuclear supernatant (SIO), polysomes or RSW as a source of messenger RNase activity. Deproteinized, exogenous mRNA can be included with any of these, as desired. (b) Various combinations of fractions, for example, polysomes or RSW plus SI30, with or without exogenous mRNA substrate, can be used. The ability to determine how various fractions influence mRNA turnover is essential for identifying and characterizing mRNases and regulatory factors (3, 6, 7). (c) With regard to possible modifications, the conditions described below should not be considered rigid (see Section 4). For example, the author has identified a mRNA destabilizer acti\ity in SI30 from cells infected with Herpes simplex "irus type I (6). The destabilizer '~as relatively inactive under the standard reaction conditions described below but was fully active in reactions containing approximately one-third lower potassium and magnesium ion concentrations. Therefore. the investigator should be prepared to modify the ~tandard system as necessary.

5 .1.1 Using endogenous mRNA as substrate The standard system for this assay is described in Protoco/6 using polysomes al> the source of endogenous mRNA. 119

4: mRNA turnover in cell -free extracts r------------------------------------------------------------~

Protocol 6. Turnover of endogenous polysomal m ANA in detergentfree extract s 8 Reagents e Solutton F. prepare this as follows and store ot on small aliquots at - 70 ° C

Component

Final concentration in 2~i 111 reaction mixtures

Ill

water

200

1 0 M creaune phosphate

200

10mM

40

2mM

1MDTI 0 . 2 M A TP (Tris salt)

100

1 mM

0 .2 M GTP (Tris salt)

20

0. 2mM

2.0 M potassium acetate

01 M

1000

2. 5 mM spermine

800

0.1 mM

1 0 M Tros- acetate. pH 7.5

200

10 mM

• Solution A and polysomes at a concentration of 106 cells' polysomes ~o~l prepared as described m Prorocol 2

solution F must be added before the RNase inhibitor; the volumes goven are for each 25 ~o~l reactoon requored in step I

• Creatine krnase (4 mg/mlon 50% glycerol)

solution F

• 0 . 1 M magnesium acetate

4 mg/ml creatine kinase

0.2 5~o~ l

• Placental RNase inhibitor (40 unots l~o~ll

RNase inhibitor

0 .25111

• Solution G prepare this solution on oce JUSt prior to performing the reactoon; the author does not know if the order of additoon of components is omportant, but

solution A

3.2~o~ l

161JI

0 . 1 M magnesium acetate

0 . 2111

water

1. 1 Ill

Method 1 . Add the following to the required number of microcentnfuge tubes on ice • solution G

21 ~o~l

e polysomes 2 . Mix gently without vonexong and incubate the reactions at an appropriate temperature, usually 37 °C , for the required t ome. No smgle reactoon temperature can be considered optimal f or all mRNA decay reactions. Therefore, while standard reactions are usually incubated at 37 ° C, Situations may arise in whoch it may be advantageous to incubat•~ at lower temperatures. For example, cell -free mRNA turnover reactions are panicularly useful for odentofying mRNA decay products. The nature of these products provides a pict ure of t he decay pathway: 1ts direction (5'- 3' or 3 ' - 5'1 and t he sons of enzymes involved (exo- or endo ribonucleases). However, decay products of some mRNAs might be too unstable to be detected even in vitro at 37 ° C . If so, try incubating the reactions at a temperature between 20 ° C and 32 ° C , rather than 37 °C . React ion rates are slower at lower t emperatures, permotting easier detection of decay products (17).

Continued

120

Jeff Ross Protocol 6. Continut?d 3 . Remove the tubes .and either store them at - 70 °C or extract the RNA immediately for analysiS. Any RNA extraction method can be used, as long as care is taken to avoid non-specific RNase contamination. RNA extraction methods are g1ven 1n Volume I, Chapter 1 From refs 13. 14.

As mentioned in Sections 4 and 5.1, various combinations of subcellular fractions can be assayed in this system, which is one of its important features. The components of the reaction mixture given in Protocol 6 (step I) can be modified accordingly. For example: (a) Some trans-acting factors that regulate mRNA stability are located in the S 130 fraction and can be ido~ntified by mixing Sl30 and polysomes. Simply substitute Sl30 for an equivalent volume of solution A when making up solution G. To assay the effect of I0 ~I of S 130 on the decay of polysomal mRNP, prepare solution G with 6 ~I per reaction of solution A, instead of 16 ~1. and set up reactions containing I 1 ~I of solution G, 10 ~I of Sl30, and 4 ~I of polysomes. (b) To analyse mRNP decay in SIO, simply substitute SIO for polysomes and solution A. Care should be taken to maintain the magnesium ion concentration at 2 mM.

5.1.2 Using exogenous RNA substrates A system for assaying the turnover of exogenous RNA in detergent-free extracts is described in Protocol 7. The method is similar to that described in Protocol 6, but the substrate is protein-free (phenol-extracted) RNA, not endogenous polysomal mRNP. The two major sources of substrate are cellular mRNA or RNA synthesized in vitro. Cellular RNA should be prepared from a cytoplasmic extract and can be pre-purified by affinity chromatography to select poly(A) + mRNA. If synthetic mRNA is used as a substrate, some important \ariables must be considered. Should the mRNA be synthesized in the presence of a cap analogue (m-GpppG)? Should it be polyadenylated? Should it be radiolabelled, and, if so, to what specific activity? As a general rule, it seems reasonabl.e initially to use a substrate as close as possible to authentic, i.e. both capped and polyadenylated. Protocol 7. Turnover of exogenous mANA substrates in detergentfree extracts 8

Reagents • SolutiOn A and polysomes 1105 cells' polysomes/IJI as described in Pro-

e creatme kinase, RNase 1nhib1tor, solutoon F, magnesium acetate as described in

toco/2

Protocol 6

Continued 121

4: mRNA turnover in cell-free extracts Protocol

7. Continued

• E•ther a ' P·•abelled RNA substrate 1- 107 c. p .m . IJQ, 5000- 20 000 c.p .m. l ~o~ l m w aterl or unlabe lled RNA 10 .1 -2.0 ~o~g . ~o~l rn water; se•:1 Volume I, Chapter 1 !Protocols 3 and 4 1 and Chapter 4, th1s volume (Pro tocol 4 I

• Solution H: prepare thrs on ice; the

formula below •s fo r each 25 ~o~l reactJOn i n step 1: solut1on F 3 . 2 IJI 4 mg ml creat1ne kmase 0 .251JI RNase mhibrtor 0 .251JI solution A 161JI 0 . 1 M magnes1um acetate 0 . 21JI RNA substrate 1 liJI reQu ~red

Method 1. Add the following to thH required number of mrcrocentrifuge tubes on ice· • solution H e polysomes

21

~I

2. Incubate the reactions at 37 °C for the des1red t ime. 3. Erther store the reaction mrxtures at - 70 ° C or extract the RNA immediately (see Protocol 6 , step 3 ). • From refs 1 3, 14.

The reaction mixtures described in Protocol 7 can be modified to incluue SIO, Sl30, or combinations thereof. When doing so, the composition of solution H must be changed, decreasing the volume of solution A o accommodate that of the added subcellular fraction. If it is necessary to add more than 2 ~g of RNA substrate per 25 ~I reaction, preliminan assays should be performed at the higher substrate levels, since excess R '
jeff Ross Regardless of the source of enzyme, it is important to ensure that the relative deca) rates observed ,.. ith endogenous substrates are maintained in reactions with exogenous substrates. As discussed in Section 2.2, exogenous substrates might fail to reflect mRNA decay processes as accuratel} as their polysomeassociated counterparts.

Protocol 8 . Turnover of exogenous mRNA substrates using ribosomal salt wash (RSW) 8

Reagents • Creatme kmase, 0.1 M magnes1um acetate. RNase inhibitor, solution A as in Protocol 6 • RNA substrate as described 1n Protocol 7

800 jJI 1 0 M Tris-acetate, pH 7 .5 200 jJI • Solution J: prepare this on ice just before

• RSW (see Protoco/3) • Solut1on I prepare th1s as follows and store at - 20 °C '" mufuple aliquots water

1.0 M creatine phosphate 1.0 M OTT

1 2 ml

2. 5 mM spermme

performing the reactions; the volumes g1ven below are for each 25 Ill reacuon requ11ed 1n step 1· solut1on I

3 .2 !Jl

4 mg ·ml creatine k1nase

200 111

RNase mh•b•tor• solut1on A

40 !JI

0. 1 M magnes1um acetate

0.2 M A TP
RNA substrate

0 . 25111 0.25111 15 jJl 0.21JI 1.1 Ill

Method 1 . Add the following to the requ1red number of microcentrifuge tubes on ice. • solution J

20 IJI

• RSW 2. Incubate the reactions at the desired temperature for the desired t1me !see

Protocol 61. 3 . Either store the reaction mixtures at immediately (see Protocol 6, step 31

- 70

°C or extract the RNA

• From ref 14. • It might be necessary to Increase the amount of RNase •nh•b•tor If so, the amount of sofut•on A should be decreased to maintain the 25 111 reaction volume

5.2 Lysolecithin extracts As discussed in Section 4, mRNA is translated more efficiently in lysolecithin extracts than in extracts prepared by lysing celJs in detergent-free buffer. ln order to exploit this property of the system, it might be useful to perform 123

4: mRNA turnover in cell-free extracts the following preliminary experimems to determine optimal translation parameters: (a) Incubate the extract at 37 oc for different times (e.g. between 30 mm and 4 h) in the presence of a radioactive amino acid (see Protocol 11 ) Determine the kinetics of incorporation of amino acids into protein by aciu precipitation. This experiment will determine the optimal time of incubation for mRNA decay assays. (b) If exogenous mRJ'lA is to be assayed in nuclease-treated extract (see Protocol 10), add 1-10 ~o~g per reaction of mRNA and measure the amino acid incorporation. This experiment is important both to determine the optimal mRNA level for translation and to ensure that reactions do not comain excess mRNA, \\hich might slow translation or mRNA decay rates , or both. (c) Having determined the optimal time and mRNA level, compare translation rates at 25, 30, and 37 °C, to optimize the temperature for translation

5.2.1 Using endogenous RNA as substrate Analysis of the turnover of endogenous mRNA in lysolecithin described in Protocol 9.

extract~ 1s

Protocol 9 . Turnover of endogenous mRNA in lysolecithin extracts8

Reagents • Post-nuclear supernatant (crude lysate) prepared as in Protocol 4

Method 1. Pipette the requ1red amount of extract I 100-200 IJil into microcentrifuge tubes on ice. 2. Incubate the reactions in a water bath at the desired temperature and for an appropnate time. 3. Either store the reaction mixtures at - 70 ° C or extract the RNA immediately (see Protocol 6, step 3). • From ref. 5

5.2.2 Using exogenous RNA substrates The use of exogenous RNA substrates with lysolecithin extracts is described in Protocol 10. See Section 5.1 .2 for a description of alternative substrates 124

Jeff Ross Protocol 10. Turnover of exogenous mANA m lysolecithin extracts Reagents • Lysolecithin extract. e1ther untreated (see Protocol 4 ) or micrococcal nuclease· treated !see Protocol 51

• Exogenous mRNA substrate in water (1- 51Jg 'llll. see Protocol 7

Method 1. Pipette 99-198 IJ) of extract and 1-21JI of mRNA substrate mto m1cro· centrifuge tubes on ice. As discussed in Section 5.2, the amount of mRNA per reaction will depend on two factors, the capacity of the system to translate a given quantity of mRNA and the possibility that excess exogenous RNA might slow translation or mRNA decay. Both factors should be investigated pnor to performing reactions. 2 . Incubate the mixture at an appropnate temperature for the des1red time. 3 . Either store the reaction m1xtures at - 70 ° C or extract the RNA immediately (see Protocol 6 , step 3).

As discussed in Section 5.1 with reference to the detergent-free extracts, reactions in ly~olecithin extracts as described in Protocols 9 and 10 may also be modified in various ways. For example: (a) The role of translation in mRNA turnover is well-documented but poorly understood (I). Therefore, the addition of reticulocyte lysate might be useful if it stimulates or prolongs translation of the mRNA under study. (b) The association of immunoglobulin mRNA with membranes might affect its half-life during B-cell differentiation (I 9, 20). Therefore, adding membranes

the system might be necessary to identify regulatory factors for certain mRNAs.

to

These possible effects can be tested by adding rabbit reticulocyte lysate and canine pancreatic membranes, both of which are commercially available (21). Reaction mixtures (see Protocols 9 and 10) would then include 200 Ill of lysolecithin extract, with or without exogenous mRNA, 20 J.JI of reticulocyte lysate, and 4 J.JI of membranes.

5.3 Reticulocyte translation extracts Analysis of exogenous mRNA turnover in reticulocyte lysate is described in Protocol II . Rabbit reticulocyte extract treated with micrococcal nuclease (obtained from a suitable commercial source) is incubated with phenol-extracted RNA from cells or from in virro transcriptions. As discussed in Sections 4 and 5.2 modifications of the reticulocyte system should be considered for particular applications. For example, Sl30, polysomes, and/ or membranes can be added. 125

4. mR.\!A turno\•er in cell-free extracts

Protocol 11. Turnover of exogenous mANA in an mANA-dependent rabbit reticulocyte translation system8

Reagents e commerc1al rabbit retiCUlocyte lysate pretreated with micrococcal nuclease (Amersham. Boehringer Mannhe1m, DuPont, Gibco BRL, Promega) • mRNA d1ssolved in water at 0 1- 5.0 11g IJI, e1ther from cells or from in vmo transcnptions. If using cellular RNA, 1t m1ght be necessary to prepare poly~!\)- mRNA. In vttro transcnpuon of RNA i:s descnbed in Volume I. Chapter 1 (Protocols 3 and 4 I and Chapter 2 , th1s volume. (Protocol 4 l

• Placental RNase tnhib1tor, 40 units/~JI (see Protocol 6 I

• 1 mM ammo acid mixture; various ammo ac1d mixtures are available from the compan,es that sell the lysate; some are complete; others lack one of the 20 amino acids, so that it can be replaced by its radioactive analogue 1n order to measure translation eH1ciency

Method Add the reagents in the order listed below to microcentrifuge tubes in an ice bath. The final reaction volume 1s 50 1-11.

351JI 1 IJI

• nuclease-treated lysate • RNase inhibitor • amino acid mixture • radioactive amino acid and/or water

1 Ill as necessary

• mRNA substrate in water

as necessary

2. Incubate at 30 °C for the required time. 3. Either store the samples at - 70 °C or extract the RNA immediately (see Protocol 6, step 31. • From refs 22, 23.

6. mRNA detection, data interpretation, and troubleshooting 6.1 mRNA detection and some typical in vitro mRNA decay

experiments Many methods are available for analysing and quantifying endogenous mR, .\ levels, including Northern blotting, nuclease Slmapping and RNase protectlo 1. primer extension analysis, and amplification by PCR. The method of chmce will depend in large measure on the quantity per cell of ~he particular mR '-A. Blotting and nuclease protection, whlcb are described in Volume I, Chapters 2. 3, and 4 and Chapter 1, thns volume, are preferred for moderately abundant mRNAs (at least 100 molecules per cell). For very scarce mRNAs, PCR 126

feff Ross amplification methodolog} might be required. Radiolabelled mR"''A substrates are the simplest to analyse, requiring only electrophoresis m a denaturing polyacrylamide gel (see Volume I, Chapter I, Protocol 8 and Volume I, Chapter 4, Protocol 11). Three examples of mRNA decay experiments from the author's laboratory are presented below to illustrate these and other points. 6.1.1 Relative decay rates of endogenous, polysome-associated

mRNPs in cell-free mRNA decay reactions The experiment shown in Figure I was performed to compare the in \1irro decay rates of three endogenous mRNAs. The experiment involved the following: • Preparation of detergent-free polysomes from tissue culture cells (human erythroleukaemia) as per Protocol 2. • Incubation of the polysomes for the indicated times essentially as described in Protocol 6. • Isolation of total RNA from each reaction by phenol extraction. • Annealing of a portion of the extracted RNA separately to each of three 32P-labelled DNA hybridization probes for human "(-globin, 6-globin, or c-myc mRNAs. • Treatment of each hybridization reaction with nuclease Sl and electrophoresis of the nuclease-resistant DNA in denaturing polyacrylamide, urea gels. • Autoradiograph} of the gels and quantification of the amount of each DNA fragment using a laser densltometer. The quantity of nuclease-resistant DNA indicates the amount of each mRNA remaining at each time point.

1: . ~-

~

~----0------0 ~.

~50

~


B. ~o

~

...

•

z 25 cr E

100

________ __ p_ -- _o

200

\

00

·~ 100

200

300

Time (min) Figure 1 Companson of in vitro stability of three endogenous. polysome-associated mANAs. Polysomes tsolated from erythroleukaem1a tissue culture cells were mcubated as descnbed in the text, and the stab1hties of three mANAs were compared by nuclease S 1 protection and Quantification of the autoradiograms. Panel A , ..,-globin mANA (unfilled circles) and 6-globin mANA (filled circles) Panel B; ..,-globon mANA (unfilled c~rclesl and c·myc mANA (foiled triangles). Aepnnted with permission from ref 1 3 .

127

4: mRNA turnover in cell-free extracts

deadenylated (j-Giobin

(j-Giobin

- PABP - PABP +PABP -PABP time 1 1 11 1 11 l minl o 20 40 0 20 4al 0 20 40 0 20 40

-

439 370-

__.H • 2 3 4

5 6

7 8

9 10 11 12 13

Figure 2. Companson of the stabilities of exogenous polyadenylated and deadenylated mANAs in ASW e1ther lacking or containing polyiA)-binding protein IPABP). ASW was first depleted of PABP It was then incubated as descnbed in Protocol 8 with either of three substrates polyadenylated I! globm mANA, I!; deadenylated f!·globtn mANA A( - )f!, or htstone mANA, H. which normally lacks poly(A) . Some reactions were supplemented wtth punfied yeast PAB?. lane 1, DNA size markers (sizes of two fragments are given tn base patrs on the left); lanes 2 -7, polyadenylated J!·globin, 1!. and deadenylated histone, H; mANAs mcubated ior the timesm
Figure I shows the decay of ;-globin mRNA, whose intracellular half-life is approximately 17 h, a-globin mRNA (intracellular half-life 4 h) and C-fll)'C mRNA, with an intracellular half-life of 0.5 h. This experiment illustrate~ an essential property of this in vitro mRNA deca) system: it reflects known intracellular mRNA half-lives. 6 .1.2 Radiolabelled substrate incubated with ribosomal salt wash The experiment of Figure 2 was performed for two reasons: (a) To compare the half-lives of two radiolabelled globin mRNA substrate~. one with an 80 nt 3 ' poly(A) tract, the other lacking poly(A). (b) To determine whether poly(A)-binding protein (PABP) influences the in vuro decay rate of either substrate. The experiment involved th~ following: 128

Jeff Ross • Preparation of three 32P-labe!Jed substrates, polyadenylated human ~-globin mRNA, deadenylated human ~-globin mRNA, and human H4 histone mRNA, by transcribing appropriate eDNA clones with SP6 RNA polymerase. Histone mRNA is one of the few mammalian mRNAs without a poly(A) tract. It sened as a control in some reactions. • Preparation of RSW from tissue culture cells (human erythroleukaemia) as per Protocol3 and then depletion of the RSW of endogenous PABP. • Incubation of substrates with RSW as per Protocol 8 for the times indicated in Figure 2. Some reactions also contained highly purified PABP from yeast. • Extraction of total RNA from each reaction and electrophoresis of each RNA in a denaturing polyacrylamide/ urea gel. • Autoradiography of the gel.

Figure 2 shows the rapid decay of polyadenylated globin mRNA and of histone mRNA in the PABP-depleted RSW. In contrast, ~-globin mRNA is at least 10-fold more stable than histone mRNA in reactions supplemented with purified PABP. Therefore, PABP contributes to the long half-life of polyadenylated ~-globin mRNA in these reactions. However, PABP does not stabilize deadenylated ~-globin mRNA. Therefore, PABP probably helps to stabilize mRNA by interacting with the 3' poly(A) tract.

6.1.3 Effect of a trans-acting mRNA stability regulatory factor in the post-polysomal supernatant (5130} The experiment of Figure 3 was performed to determine whether the destabilization of ,-globin and other host cell mRNAs following Herpes simplex virus infection could be reproduced in vitro and, if so, in \\hat subcellular fraction the destabilizing factor resided. The following experiment was performed: • Two sets of tissue culture cells were used. One was infected with Herpes simple>.. \irus, the other was 'mock infected'. meaning that it was treated in the same way but no virus was added. • At 60, 180, and 360 min following virus inoculation (mixing virus and cells). cells were harvested and post-polysomal supernatant (S 130) "as prepared. as per Protocol 2.

• In vitro mRNA decay reactions were performed by mixing polysomes from uninfected cells wnh Sl30 from either mock-infected or "irus-mfected cells, as per Protocol 6. • Each reaction was incubated for 0 to 150 min; then total RNA was isolated and annealed with a 31P-labelled DNA probe for human -y-globin mRNA. The amount of undegraded ,-globin mRNA in each reaction was determined by mcubation with nuclease Sl and electrophoresis of nuclease-resistant DNA in a gel. 129

4: mRNA turnover in cell-free extracts

Mock in V//ro

0 15 30 90 150

Incubation T1me (min)

Y-Giobin-.

Wlld-type Post-Inoculation Time(min)

180 o

15 30 90 150 0

15 30 90 150 0

360 15 30 90 150

In vitro

< lncubat1o1 nme(m•

~

Y-Giobin -.

Figure 3. Identification of an mRNA destabillzmg factor 1n the S 130 of cells mfected w1th Herpes s1mplex v1rus. Tissue culture cells were either mock -infected or infected with Herpes Simplex virus for up to 6 h, as described in the text. S 130 was prepared from each culture flask and was added to polysomes from uninfected cells. The react1ons were Incubated as per Protocol 6. The stability of polysome-associated -y-glob1n mANA was determined by nuclease S 1 mappmg. Top panel, polysomes plus S 130 from mock-infected cells, 1 e. cells not exposed to virus. Bottom panel, polysomes plus S 130 from cells infected for the indicated times [wild-type, post-moculation time (min) I with Herpes s1mplex v~rus . The time course 1n mm of each incubation is shown {in VItro •ncubation time lmml I above each lane. Repnnted with permiSSIOn from ref. 6

Figure 3 shows that polysomal, endogenous )-globin mRNA is ver} stable in reactions supplemented with SJ 30 from mock-infected cells (no virus) but that S 130 from cells infected with the virus for 60, 180, or 360 min contains a potent mRNA destabilizer. Th.is destabilizer is now knO\\n to be a protein encoded by the' irus and an integral part of the virion itself. By an unknown mechanism. this protein accelerates the degradation of almost all cellular mRNAs, even tho,e like )-globin which are normally very stable in uninfected cells.

6.2 Data interpretation As discussed in Section I and Protoco/6, mRNA decay rates are slower in ,.;rro than in intact cells. Relatively slow reaction rates are also observed in cell-tree 1 30

Jeff Ross transcription and translation systems. Therefore, it seems reasonable to exploit cell-free mRNA decay systems to measure relative, as opposed to absolute, decay rates. To assess whether the system is functioning properly, the decay rates of mRNAs which are known to have different intracellular half-lives should be determined. mRNAs that are unstable in ceiJs should also be unstable in vitro, relathe to stable mRNAs. Some caution must be exercised in plotting and interpreting in vitro mRNA decay data. In cells, mRNA is degraded in a logarithmic fashion, according to the formula:

where C and C 0 are mRNA levels at time t and time 0, respectively, and kd is the decay constant (reviewed in ref. 25). The problem with applying similar parameters to in vitro data is that decay rates may decrease as the reaction time increases. For example, the author has found that the c-myc mRNA decay rate decreases at least two-fold after several hours of incubation, presumably because critical reaction components become rate-limiting (J. Ross and G. Brewer, unpublished observations). Therefore, it seems reasonable to plot the in vitro data as either a linear or logarithmic function , provided that each mRNA is analysed by the same criteria (see Figure I as an example).

6.3 Troubleshooting Several potential obstacles and trouble spots which might arise during the course of cell-free mRNA decay experiments are listed below, as well as some suggestions for solving them.

6.3.1 Excessive RNA degradation during preparation of polysomes and extracts Precautions for reducing endogenous, non-specific RNase activity while preparing polysomes and during in vitro incubations are discussed in Protocol 2. If these are inadequate, it might be helpful to avoid using primary animal ceiJs and to use instead a tissue culture cell line synthesizing the mRNA{s) of interest. Some cells lines contain less 'non-specific' nuclease activity than others. Try screening several ceiJ lines to find one which allows intact polysomes to be isolated.

6.3.2 All mRNAs in the mRNA decay reactions are degraded at similar rates Again, non-specific RNase activity might be overwhelming the system. Se"eral modifications should be tried: • Add more RNase inhibitor or try various combinations of inhibitors. 131

4: mRNA turnover in cell-free extracts • Modify the reaction conditions. For example, non-specific RNase acti\ il} might be depressed by changing the mono' alent or divalent cation concentrations, the pH, or the reaction temperature. • Add carrier single-stranded RNA, for example, poly(C) at 1-21Jg 25 ~ reaction (see Protocols 6-8). Avoid higher amounts of exogenous RNA, whkn can slow mRNA decay processes.

6.3.3 No mRNA degradation is observed The simplest explanation for this result would be inactivation of messenger RNases (and other RNases) during extract preparation. If no RNA is degraded during in vitro reactions, the composition and pH of all extraction buffers should be monitored carefully. An unlikely explanation is that all RNase activity tn the extract is blocked by the placental RNase inhibitor. Therefore, reactions should be carried out without inhibitor.

6.3.4 Regulated decay is not observed For example, a particular mRNA might be known to be degraded five-fold It ~s rapidly in cells treated with a translational inhibitor, as compared with untreated cells. However, the mRNA is found to be degraded in vitro at the same rate with polysomes isolated from treated and untreated cells. There could be many explanations for this result, but the following can be investigated: • Add S 130 (Protocol 2) to the reactions. Perhaps the regulatory factor is n the S130 and is not polysome-associated. • Alter the reaction conditions, as per Section 6.3 .2. • Prepare a lysolecithin extract (Protocols 4 and 9) rather than a detergentfree extract from both sets of cells, to encourage translation of the mRN A., since the regulation of some mRNAs might be linked to their translation. In this case, the mRNA might be unstable under conditions of efficient translation elongation, but stabilized when elongation is retarded by an inhibitor. The in vitro system would then reflect the intracellular situation only by comparing the stability of elongating versus non-elongating mR}'I. '\, Some translation does occur in the lysolecithin extract but little is obsen t.'
References I. Peltz, S. W., Brewer, G., Bernstein, P., Hart, P. A., and Ross, J. (199l).ln Criflcal reviews in eukaryoric gene expression (ed. G. S. Stein, J. L. Stein, and J. B. Lian )

Vol. I, p. 99, CRC Press, Ann Arbor. 2. Belasco, J. G. and Higgins, C. F. (1988). Gene, 12, 15. 3. Brewer, G. and Ross, J. (1989). Mol. Cell. Bioi., 9, 2006.

132

Jeff Ross 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25.

\\ager, R. E. and Ass01an, R. K. (1990). Mol. Cell. Bioi.• 10, 5893. Krikorian. C. R. and Read, G. S. (1990). J. Virof., 65, 112. Soren~on. C. \I., Han, P. A., and Ross, J. {1991). ,...ucf. Acids Res., 19,4459. Brewer, G. (1991). Mol. Cell. Bioi., 11 , 2460. Blume, J E. and Shapiro, D. J. (1989). Nuc/. Acids Res., 11, 9003. Graves, R. A., Pandey, 1'\. B., Chodchoy, N., and Marzluff, \v. F. (198.,). Cell, 48, 615. Capasso. 0., Bleecker, G. C., and Heintz, N. (1987). EMBO J., 6, 1825. Blackburn, P. and Jailkhani, B. L. (1979). J. Bioi. Chem., 254, 12488. Berger. S. L. and Birkenmeier, C. S. (1979). Biochemistry, 18, 5143. Ross, J. and Kobs, G. (1986). J. Mol. Bioi., 188, 579. Ross, J., Kobs, G., Brewer, G., and Peltz, S. W. (1987). J. Bwl. Chem., 262,9374. Bradford, M. (1976). Anal. Biochem., 72, 248. Brown, G. D., Peluso, R. W., Moyer, S. A., and Moyer, R. W. (1983). J. Bioi. Chem .• 258, 14 309. Peltz, S. W. and Ross, J. (1987). Mol. Cell. Bioi., 7, 4345. Peltz, S. W., Brewer, G., Kobs, G., and Ross, J. {1987). J. Bioi. Chem., 262,9382. Mason, J. 0., Williams, G. T .. and Neuberger, M.S. (1988). Genes Develop., 2, 1003. Jack, H-\1. and Wabl, M. {1988). EMBO J., 7, 1041. Baker, S.C. and Lai, t-1. M. C. (1990). EMBO J., 9, 4173. Wreschner, D. H and Rechavi, G. (1988). Eur. J. Biochem., 172, 333. Hepler, J. E., VanWyk, J J. , and Lund, P. K. (1990). Endocrinology, 121, 1550. Bernstein, P., Peltz, S. W., and Ross, J. (1989). Mol. Cell. Bioi. , 9, 659. Hargrove, J. L., Hulsey, M.G., and Beale, E. G. (1991). BwEssays, 13, 667.

133

Ribosomal RNA processing vertebrates

.

lll

CATHLEEN ENR IG H T and BARBARA SOl LNER-WEBB

1. Introduction About half of the transcription of an actively growing eukaryotic cell is devoted to the production of the large ribosomal RNA precursor (47S or - 14kbp in mammals) that gives nse to the 18S, 5.8S, and 28S mature rRNAs of the ribosome (see Figure 1 ). Thus a major processmg commitment of the eukaryotic cell is de\oted to the maturation of rRNA. This rRNA processing occurs in the nucleolus and involves: • cleavages at the 5 '-and the 3 '-ends of each of the mature rRNA segments • cleavages at a still undetermined number of sites within the transcribed spacer regions • numerous methylation events D ue to the large cellular abundance of the mature rRNAs and a number of rR NA processing intermediates, the cleavages that give rise to the 5'- and the 3 '-ends of the ISS and 28S rRNAs were able to be identified prior to the recombinant DNA re\olution, making rRNA processing the first eukaryotic transcript maturation pathway in which the major stages were described. Despite this early progress, a great deal still remains to be learned about rRNA processing. For a recent revie.,., of the field of eukaryotic rRNA processing, see ref. I. This chapter focuses on rRNA processing in venebrates and describes laboratory techniques available for its experimental investigation. Since most is known about the primary processing event that occurs within the 5' external transcribed spacer of mouse rRNA (5' ETS, see Figure 1), most attention will be given to: • re-creation of this processing event in vitro using synthetic r RNA substrates and cell extracts possessing processing activities • identification of the processing signals within the rRNA

5: Ribosom al RNA processing in vertebrates PROCESSING OF VERTEBRATE PRE-rRNA

rrs 1 rrs 2

5' ETS

•

3'ETS 47

16$

S' ETS pr0C8SS1ng

5.6$

s

26$ 46S

45S

/

--

16S

368

16S

32S

16 s

\

41 s

alternative

32 s

34S

-

20S 26S

"-

16S

32S

I •

26S

5.6S

Figure 1. Diagrammatic representation of rRNA processing in vertebrates. The compos,te dragram draws on data from mouse, human, frog, and other vertebrate species: the sedimentation coefficients of the processing intermediates are shown for mouse rRNA Two alternative pathways are shown. There may well be other minor pathways and it rs not certatn whether every rRNA precursor must pass through each of the processing steps in the order shown. 5' ETS, 3' ETS; external transcribed spacer regions at the 5'- and 3'-ends of the rRNA transcnption unit, respectively ITS 1 and ITS 2; internal transcribed spacers . The arrow head shows the position of the cleavage site in the 5' ETS referred to in the text. !Adapted f rom ref. 1; where further information can be found. )

• analysis of the nucleoprotein complex that forms on the processing signal • identification of the polypeptides that bind specifically

to

this rRNA region

• identification and analysis of the requirement for U3 snRNP in this processing reaction Recem evidence indicates that such a 5 1 ETS processing complex is found in many if not all eukaryotes. Relatively little is known about most of the other conserved eukaryotic rRNA processing events, with the exception of the processing of the 5 -end of the ISS rRNA region. This 5 ' processing has been reproduced in vitro as a two step (cleavage and trimming) maturation, as 1

136

Cathleen Enright and Barbara Sollner-Webb Table 1. Metazoan rRNA processing events currently accessible to study in systems amenable to experimental manipulation Processing event

Species

Method of study

5 ' ETS cleavage

mouse human Xenopus mouse

in S-1 00 extract of mouse cells in S-1 00 extract of mouse cells in S- 1 00 extract of human or mouse cells using nucleolar endonuclease

human mouse mouse

in nucleolar extract of human cells in nucleolar extract of mouse cells by transfection into rat cells

12

mouse

using nucleolar endonuclease

14b

by following oocyte endogenous rRNA processing in mouse cell extracts

16 17

by oocyte injection

18

Cleavage at 5 '-end of 18S region

Cleavage at 3 '-end of 18S region

Cleavage at 5'-end of 5.85 region Xenopus mouse termination-associated 3'-end processing Xenopus

Reference 9 , 10 10 40 11

13

•r . Nilsen, personal

communication. • But see reservation in ref. 15.

described in Section 3.3. Table I summarizes the major metazoan rRNA processing events whose study has thus far been reported. This chapter does not cover: • genetic techniques that are used to analyse rRNA processing in yeast (2-6) • the removal of the rRNA intron that is peculiar to certain unicellular eukaryotes (see Chapter 7) • the cleavages that yield fragmented rRNA in certain species (described and reviewed in ref. 7) • processing of nucleolar small nuclear RNAs (reviewed in ref. I) • analyses of rRNA methylation (reviewed in ref. 8)

2. Analysis of rRNA processing in vivo 2.1 Introduction Clearly, the first step in analysing an RNA processing event is to define the process in vivo. Significant data on rRNA processing have been derived through the identification of processing intermediates and mature rRNAs by size and structural features, thereby allowing processing sites to be located within the primary transcript and processing steps to be temporally ordered. The early 137

5: Ribosomal RJ\A processing in vertebrates analy~es

of processing involved lractionation of rRNA b~ \elocity gradient ultracentrifugation and the electron microscopic analysis both of the secondar• structures of rRNA~ and of their heteroduplex formation with cloned rRNA genes (see refs 19-21 for procedures). The major vertebrate rRNA processmg intermediates and mature rRNAs were thus deduced to be cleavage fragmen of the primal] transcript, without the discontinuitie:. that \\Ould anse b} splicing RNA editing, etc. This conclusion has since been confirmed by nuclease 5 mapping and nucleotide sequence analysis. These early studres also showed that there is a preferential but not obligatory order to the cleavages at the ends of the mature rRNA regions (see Figure 1).

1 2 3

47S,

46 s-

rt

45S"

32S ...

285

Figure 2. Gel electrophoretoc analysis of the early processong reactions of mouse pre-rRNA RNA was labelled in vivo by growing mouse tissue culture cells for 1 h on the presence of 3 [ Hlurodine. RNA was then extracted and fractionated by hybnd selection using a cloned rONA segment correspondong to residues 1-292 of the rRNA uanscroptoon untt flane 2). or to the 282 nt sequence that begons 58 nt beyond the 3 '-end of the 285 rRNA regoon (lane 31 Unfractionated rRNA is shown in lane 1 Following electrophoresos, ( 3 HlRNA was detected bv fluorography. Res.dues 1-292 of the 5' ET5 are present only on the largest rRNA 14751 wherea~ sequences of the 3' ET5 are present on both the 4 75 and 465 species but not in smaller rRNAs such as the 325 rRNA (see Protocol 1 for details I Adapted from ref 22 w1th permossoon.

138

Cathleen Enright and Barbara Sollner-Webb

2.2 Analysis ofrRNA processing by hybrid selection and gel

electrophoresis of cellular rRNAs Precursor RNA and processing products are now generally analysed by gel electrophoresis rather than by ultracentrifugation. In a striking application of this, the initial processing events in mouse rRNA maturation have been ordered temporally b}' electrophoretic sizing of different populations of rRNA intermediates, isolated by hybrid selection (22). This approach is illustrated in Figure 2 where the data show that the cleavage within the mouse 5' ETS precedes the removal of the 3 ' ETS, which in tum occurs prior to the cleavages at the 5 ' -and 3 ' -ends of the 18 and 5.8S regions and at the 5' -end of the 28S region (within the 45S rRNA; see Figure 1). Protocol 1 describes the procedure for labelling rRNA in vivo, the isolation of rRNA processing intermediates by hybrid selection and their separation by analytical gel electrophoresis. An alternative approach would be to use unlabelled rRNA and then detect the rRNA intermediates after electrophoresis by Northern bloning with appropriate rRNA probes. Procedures for Northern blotting and probe preparation are given in Volume I , Chapter 2 (Section 3.6), Volume I, Chapter 4 {Section 7.5), and ref. 23.

Protocol 1 . Analysis of rANA intermediates by hybrid selection and gel electrophoresisa Eqwpment and reagents • SV 40-transformed Balb/c mouse cell line SVT2

• vacuum dot blot apparatus (Schleicher and Schuell)

• Growth medium (DMEM with 5% newborn calf seruml and tissue culture dishes (8 cm 2 or 20 cm 2 )

• Nitrocellulose !Schleicher and Schuelll

3

• (5- H] und1ne 11 B Ci/mmol, 1 mCi/ml, ICNI or I5.6-3 H I uridine (40 Ci/mmol, 1 mCi1ml. ICN1 • PBS 120 mM potassium phosphate. pH 74. 0.13 M NaCil • Lys1s buffer (0.15 M NaCI, 50 mM TnsHCI, pH 8 1, 25 mM EDTA. 0 . 5% SDSI • Pronase IS 1gma 20 mg ·ml in 10 mM Tns-HC pH 7. 5 10 mM NaCil Selfdigest the pronase by incubatmg the solution at 37 °C for 1 h. Store the solution 1n aliquots at - 20 °C • Phenol (redistilled, buffer saturated) chloroform (1 1 vlvl • Hybnd1zauon butter (0. 4 M NaCI 0.1 M Pipes-KOH , pH 6.8, 10mM EDTA. 70% v/v formamide0)

• Denaturation buffer (50% VIV formam•de0, 2 .5 mM EDTA. 2M formal de hyde, 40 mM triethanolam1ne-HCI, pH 7. 51 • 1o x ssc (1.5 M NaCI, 0 . 15 M tnsodium citrate) • Plasmid DNA contaimng the des1red rONA sequence, linearized at an appropriate restriction enzyme s1te D1ssolve 100 1-19 linearized DNA 1nto 2 ml of denaturation buffer and incubate 11 at 55 °C for 15 m•n Dilute the denatured DNA to 2 IJQiml in 10 X sse. Store It at -20 °C •Punch made from sharpened stemless· steel tubmg (internal d1ameter 3 mm corresponding to the wells of the dot blot apparatus) •Glass fibre filters (Whatman GF/C)

Continued

139

5: Ribosomal RNA processing in vertebrates

Protocol 1. Continued • Filter holder (Hoefer) • Low-salt wash buffer. 10 mM Tns-HCI, pH 7 5, 1 mM EOTA, 0 . 1% !w 'WI SDS • End-over-end rotating wheel shaker • Modrfied Oenhardt 's buffer: 0 . 15 M NaCI, 30 mM Tris- HCI, pH 7.5, 2. 5 mM EOTA 25 mM sodium phosphate, 0 . 22 mM sodium pyrophosphate. 25 ~g/ml sonrcated, heat-denatured salmon sperm DNA, 1 mg/ml Ficoll 400, 1 mg ml dextran sulphate 500 000. 1 mg ml BSA, 1% (w/ w l SDS Prior to use, inactivate the RNase acuvrty in Denhardt' s buffer by stirring the buffer w ith 0 . 5% DEPC. Destroy the DEPC by tncubatron at 90 °C for 20 min. After cooling, adjust the pH to 7. 2

• Elution buffer (91 % v /v formamrde0 , 30 mM Pipes-KOH, pH 6.8 , 2 mM EDTA) • Honzontal submanne gel apparatus for agarose gels ( 14 em long x 19 em w ide x 0 .4 em deep) • Concentrated formaldehyde !37 % -12.3 Ml; store in a fume hood • Formaldehyde electrophoresis buffer !2 .0 M formaldehyde. 2 mM EDTA 40 mM triethanolamine-HCI, pH 7 . 51 • Reagents for fluorography (e.g. Kodak GBX film, X-ray f ilm developer, Kodak rapid hxl

Method

NB Formaldehyde is toxic. Store and use all solutions containing formaldehyde in a fume hood. Labelling of cells and extraction of RNA

1 . Seed the SVT2 cells in 8 cm 2 or 20 cm 2 tissue culture dishes at 2 x 105 cells/cm 2 and grow them as monolayers in growth medium at 37 °C in 95% air: 5% C02 for 18-24 h until the cells are in mid-log phase. 2 . Add 3 H-Iabelled uridine precursor to each dish (20 ~Ci per 20 cm 2 dish or 10 ~Ci per 8 cm 2 dish) and incubate for an additional 1- 2 h. 3 . Chill the cultures rapidly to minimize RNA degradation. To do this, decant the growth medium and replace it (within 2 sec) with 5 ml of ice-cold PBS Rinse the cells twice more (5 sec per rinse) with ice-cold PBS.

4 . Lyse the cells by adding 1- 2 ml of lysis buffer containing 100 ~g lml self digested pronase (i.e. 5 or 10 Ill of 20 mg/ml stock) at 20 ° C and incubate the cells at 20 °C.

5 . Scrape the lysed cells into a test tube and incubate the lysate for 30 mrn at 45 °C.

6 . Extract the lysate by vortexing with an equal volume of phenol:chloroforrn at room temperature . 7 . Separate the phases by centrifugation for 2 min at 10 000 g . Add 2. 5 vol of absolute ethanol to the aqueous phase and incubate the mixture in ar ethanol ice bath for 1 5 min. 8 . Collect the RNA preciprtate by centrifugation at 12 000 g for 20 min at 4 °C . 9 . Wash the RNA pellet by resuspending it in 70% ethanol and centrifuging for 1 mrn (as in step 8).

Continued 140

Cathleen Enright and Barbara Sollner-Webb Protocol 1 Continued 10. Dry the RNA pellet under vacuum and resuspend it at - 1 mg/ml in hybridization buffer The specific radioactivity of the RNA should be -1 x 105 c.p m. 'IJg. Hybrid selection using DNA nttroce/lulose filters 11 . Assemble the dot blot apparatus with a sheet of nitrocellulose accordtng to the manufacturer' s instructions. 12. Apply 0 . 5ml aliquots of the linearized denatured DNA (21Jg ml rn 10 x SSC) to the wells of the dot blot apparatus. Apply a slow vacuum to draw the solution through the nitrocellulose. 13. Dismantle the apparatus. Dry the filter under vacuum and bake it for 2.5 h at 80 °C in a vacuum oven. 14. Usrng the starnless-steel punch, cut out ctrcles (3 mm in diameter) from the nitrocellulose sheet corresponding to the posrtions of thl! wells of the dot blot apparatus. 15. Incubate each crrcle of DNA-nitrocellulose with 51.11 of RNA solution (from step 10) for 3 h at 52-54 ° C in a sealed microcentrifuge tube. 16. After hybridization, wash the filters in four successive steps to reduce background: {a) Low-salt wash on GF C filter. Remove the filter from the tube (step 1 5) wrth forceps and bend the filter with the forceps to increase washing efficiency. Place 1- 5 bent filters on a 2.5 em What man GF/C fibre-glass filter lying on the filter holder. Rrnse the GF/C: filter 20 times with low-salt wash buffer (5 ml each time). (b) Low-salt rotary wash. Remove the DNA filters from the GF/C filter. Place them in a test tube with 0.5 ml per filter of low-salt wa1sh buffer. Cap the tube and rotate it for 1 h on the end-over-end s.haker. (c) Denhardt 's 55 ° C rotary wash. Place the DNA filters in a test tube with 0 . 5 ml per filter of modified Denhardt's buffer. Cap and rotate for 2h at 55 °C. (d) Rehybridization . In a clean microcentrifuge tube, rinse the DINA filters twice with 0.5 ml of low-salt buffer per filter and once with 0. 1 ml of hybridization buffer per filter. Centrifuge the tube briefly and remove the hybridization buffer. Add 2 1.11 per filter of fresh hybrrdization buffer and heat it for 10 min at 70 °C. Then n~hybridize the filters for 2 h at 49 °C with rotation. 17. Elute the rRNA from the washed filters at 90 °C for 10 mrn in 101.11 of elution buffer. Formaldehyde/agarose gel electrophoresis 18. Cast a horizontal 0. 7% agarose gel in formaldehyde electrophoresis buffer. NB Formaldehyde is toxic. Cast the formaldehyde gels and run them in a fume hood.

Continued 141

5: Ribosomal RNA processing in vertebrates Protocol 1. Contmued 19. Bnng the eluted RNA (step 17) to 55 °C and add concentrated formaldehyde to a final concentration of 2 M Incubate the RNA for 15 mm at 55 °C. 20. Load the RNA on to the 0. 7% agarose gel and electrophorese the gel for 650-1000 volt -hours at room temperature usmg electrophoresis buffer.

21 . Impregnate the gel w1th an enhancer fluor, us1ng the method recommended by the manufacturer 22. Visualize the RNA bands by autoradiography. • From refs 22 and 24. • Deoonize the formamtde us1ng a moxed bed ion exchange reson. However, there is no need to deoonize BRL Ultrapure formamide before use. Store the formamide at - 20 °C

2.3 Use of psoralen cross-linking for the analysis of associations between rRNAs in vivo Cross-linking with psoralen has been successfully employed to investigate the potential juxtapositioning of two r RNA species during processing, by creating covalent cross-links bel\\ een base-paired or otherwise closely-positioned nucleotide residues. This technique is a powerful one for the characterization of RNP complexes, because it allows detection of RNA associations that are stabilized by proteins as well as ones that are formed directly between RNA~ Psoralen cross-linking has been used to show that the human and rat U3 snRt-.As are specifically associated with the pre-rRNA within the few hundred nucleotides surrounding the 5' ETS processing site (25, 26). The U3 sequence that is cro~s­ linked to the rat 5' ETS was then identified by reverse transcribing the cros~­ linked RNA. The transcriptase is unable to transcribe past the cross-linked nucleotides (see ref. 26 fo r procedures). Protocols for cross-linking and for the analysis of the cross-linked spec1es are described in Volume I, Chapter 5 (Section 7) and refs 25 and 26. However Protocol 2 brieOy describes a procedure for in vivo cross-linking with psoralen using rat hepatoma cells. This procedure can be adapted easily for an alternath e cell line.

Protocol 2 . Analysis of snRNA and rRNA interactions by psoralen cross-linking in vivo Equipment and reagents • Rat Novikoff hepatoma cells. (HGPRT , NISI 671

• PBS (see Protocol I )

• Growth medium tDMEM With 10% foetal calf serum and 2 mM glutamone)

• TS buffer tO 15M NaCI, 10 mM TrisHCI pH 7 5)

Continued

142

Cathleen Enright and Barbara Sollner-\\'ebb Pro tocol 2.

Continued

e AMT psoralen (HRI Associates. Inc.) dissolved in TS buffer

• Proternase K

• Long-wave 365 nml UV transrtlumrnator (e.g. Model C-62, Ultra-Violet Products, Inc.)

• Phenol, buffer saturated

e TE-SDS buffer (10 mM Trrs-HCI, pH 7.2, 5 mM EDTA, pH 8 0 , 0 . 1% SDSI

• Phenol:chloroform:rsoamyl alcohol (25 24: 1 v/V) • 4 .0 M sod rum acetate. pH 5.4

Method 1. Grow the cells to a dens1ty of 0. 5-1 x 106 cells/ml 2. Rinse the cells tw1ce w1th PBS, - 5 ml each rinse. 3. Resuspend the cells

1n

0.1 vol. of 1ce-cold TS buffer.

4 . Add AMT psoralen (final concentration 20 ~g/mll 5 . Place the cells 1n an open Petri dish on ice 6. Irradiate the cells for - 1 5 min (in 5 min intervals cooling on 1ce in between) using 365 nm light from a long-wave UV transilluminator at 1700 ~W cm 2 • 7 . Briefly pellet the cells in a m1crocentrifuge. Remove the buffer 8 . Resuspend the cells in TE-SDS buffer. 9

Pass the suspension twice through a 23-gauge needle.

10. Add proteinase K to 0.4 mg. ml and digest the cells for 2-3 h at 65 °C, with occasional gentle mixing. 11 . Extract the mixture twice with phenol and once w1th phenol:chloroform· isoamyl alcohol. In each case separate the phases by centrifugation at 10 000 g for 2 min Remove and retain the upper (aqueous) phase. 12. Add 0.1 vol. of 4.0 M sodium acetate, pH 5.4. 13. Add 1 vol. of absolute ethanol. 14. Spool out high molecular weight DNA and d1scard 1t. Precipitate the remaining nucleic ac1d with the addition of a second volume of ethanol. 15. Collect the precipitated RNA by centrifugat ion at 10 000 g for 10 min. 16. Determine t he position of the UV cross-link by pnmer extens1on of the purif1ed rRNA (step 15) with reverse transcnptase and the appropriate 5 end-labelled oligonucleotide pnmers and analys1s of the RNA on denaturing polyacrylamide gels (see Volume I, Chapter 1, Section 2.3; Chapter 2, Section 3 5; Chapter 4 , Sect1on 7 2).

2.4 Electron microscope analysis of rRNA synthesis

(Miller spreading) Another valuable technique for analysi ng rRNA processing in vivo is the ch romatin spreading and electron microscopic visualization method pioneered 143

5: Ribosomal RNA processing in \'ertebrates

by Oscar Miller's laboratory (27) over two decades ago. These imestigators demonstrated that actively transcribed rRNA genes appear as structure~ that have been called 'Christmas trees', with the DNA axis forming the trunk of the tree, the nascent rRNA molecules and associated proteins forming the branches, and a biochemically undefined 'terminal knob' forming a ball at the 5' -end of each branch. Recent work (reviewed in ref. 41) has shown that this 'terminal knob' is the processing complex that forms at the 5' ETS processing site. Spliceosome complexes have also been visualized on nascent pre-mRNAs by Miller spreading (28). While far from being simple protocols (27, 28), for those skilled in electron microscopy this spreading technique is a very powerful method for studying processing because 11 provides spatial and temporal data that are not readily obtained from biochemical analyses.

3. Analysis of rRNA processing in vitro 3.1 Introduction In order to study the events of rRNA processing, it has proved extremely advantageous to have a system in which the processing can be re-created in virro, using fractionated processing components and synthetic rRNA substrates. Processing extracts have been relatively easy to obtain from mouse and Xenopus for the 5' ETS cleavage reaction, and the reactions that excise the ISS region have also been amenable to in vitro analysis. The other metazoan rRNA processing events, however, have thus far been refractory to re-creation in vitro. Table I summarizes the available systems for in vitro analysis of processing at recognized metazoan rRNA processing sites. In yeast, the processing that occurs at the 3 ' -end of the 25S region has been re-created in vitro using a whole cell extract and syntheti.. substrate rRNA (29). An alternative approach has been to study th~" processing of rRNA transcripts in yeast transformed by the introduction of rRNA genes that have been 'tagged' by the introduction of heterogenou~ sequences (2, 3).

3.2 Preparation of synthetic rRNA substrates Substrate rRNAs for m vitro rRNA processing are most commonly ~ymhesized using a bacteriophage RNA polymerase and a plasmid template in which the desired rRNA gene region has been cloned adjacent to the corresponding bacteriophage promoter (ref. 23 and Volume I, Chapter I, Section 2.2 and this volume, Chapter 2, Section 2.6). Prorocols 3-5 describe the synthesis of radiolabelled rRNA substrates using then phage promoter and T7 RNA polymerase. 144

Cathleen Enright and Barbara Sollner-\Vebb Protocol 3 . In vitro preparation of internally-labelled rRNA substrate using T7 RNA polymerase"

Equipment and reagents • Reagents for in vttro synthesiS of radiolabelled rRNA substrates usmg T7 RNA polymerase and rONA template (see Volume I. Chapter l Protocol 4 and Chapter 2, th1s volume, Protocol 41 • Denatunng polyacrylamide (sequencmg) gel, reagents and equipment (Volume I, Chapter l, Protocol 8 or Volume I, Chapter 4, Protocol 11) e [a- 32P]CTP (ICN; 800 C1/mmol, 10JJCi/JJI) or [5- 3 H]CTP (ICN; 25 Cil mmol, 10 JJCi/JJI)

• Gel elution buffer (0.5 M ammonium acetate, pH 6 .5, 0 1% SDS, 0 . l mM EDTAI • Glycogen !molecular biology grade; Boehnnger Mannhe1ml

eo 25M

NaCI

• D1ethylpyrocarbonate (DEPCHreated H,O. add DEPC to 0.01% (v/v) and allow the water to stand for 1 h , then autoclave 1t to inactivate DEPC

Method 1. Synthesize rRNA regions of interest usmg T7 RNA polymerase as descnbed m Volume I, Chapter 1, Protocol4 or Chapter 2, this volume, Protocol4, in the presence of 800Ci/mmol [a-32P]CTP and 230 ng plasmid rONA template per 40 IJI reaction. To synthesize larger amounts of longer-lived substrate that is not 32 P-Iabelled, use 25 Ci/mmol [ 5-3 H ) CTP and 1 IJg plasmid rONA template per 100 JJI reaction. To determine the yield of RNA (see below) count a 2 JJI aliquot and calculate the tota l radioactivity. 2 . Fractionate the rRNA by electrophoresis on a sequencing gel (Volume I, Chapter 1, ProtocolS or Volume I, Chapter 4 , Protocol 11) and v1sualize the 32P-Iabelled bands by a 2 min exposure to X-ray film. 3 . To 1solate the RNA, cut the appropnate band from the gel and crush the gel slice in a microcentrifuge tube using a disposable pipette tip. Incubate the crushed gel slice in 0 . 5 ml of gel elution buffer contammg 20 IJg glycogen for at least 2 h at 37 °C. 4. Add 2.5 vol . of ethanol and collect the precipitate !Protocol1, steps 7-9). 5 . Resuspend the rRNA in 0.25 ml of 0.25 M NaCI by vortexmg vigorously. 6. Centrifuge the suspension at 1 2 000 g at room temperature for 10 min and again ethanol precipitate the rRNA from the supernatant (step 4). 7 . Wash the rRNA pellet in 70% ethanol and resuspend 1t in 50 IJI of OEPCtreated H 2 0 . Determmation of the RNA yield 8. Calculate the total input c.p.m. of CTP (or other labelled nucleotide as appropnate) from the total radioactivity in the transcription reaction !see step 1 above).

Continued

145

s·

Ribosomal RNA processing in \·ertebrates

Protocol 3. Continued 9

To calculate the specific activity of the CTP used in the reaction (c p. m pmol) div1de the total rad1oact1v1ty (step 81 by the number of pmol of CTP in the reaction (pmol of CTP =~M CTP in the reaction multiplied by the volume in m1crolitres of the reaction).

10. Count an aliquot of the purified RNA (from step 7) and calculate the total c.p.m in the isolated ANA .

11

Dtvtde calculation 10 by calculation 9 to calculate the total number of pmol of CTP Incorporated mto the 1solated RNA

12. To calculate the pmol of isolated RNA, div1de calculation 11 by the number of CMP residues in the RNA sequence Steps 2-7 are adapted from ref. 30.

Protocol 4. In vitro preparation of 5 end-labelled rRNA substrate

Equipment and reagents • Reagents for in v1tro synthesis of unlabelled rRNA substrate using T7 RNA polymerase and rONA template (see Volume I Chapter 1 Protocol 31

• 3.0 M sod1um acetate , pH 5.2

• Calf mtesunal alkahne phosphatase ICIP) IGibco-BRL, 24 unttSIIJ()

• 10" kmase buffer (0. 5 M Tus-HCI pH 7 5. 0.1 M MgCI2 • 50 mM OTT 1 mM EDTA, pH 8.01 or 10 x One·Phor· All Buffer Plus (Pharmac1a)

• React 2 buffer (Gibco-BRL 50 mM TnsHCI, pH 8 .0, 10 mM MgCI2 • 50 mM NaCI) • Denaturing polyacrylamide (sequencmgl gel, reagents for gel isolation of RNA . and DEPC·treated H 2 0 as for internally· labelled substrates lsee Protocol 3 above) e o. 1M EDTA e 20% SDS e 10 mglml proteinase K

• Phenol: chloroform ( 1 1, vlv) • 0. 2M OTT, store at -20 °C

• 1~32PJATP (ICN; 3000CLmmol.

10tJCI'IJII e TE buffer 110 mM Tris-HCI, pH 8.0. 1 mM EDTA) • 1 .0 mg ·ml BSA IBRL, acetylated BSA diluted in TE buffer) • T4 polynucleotide k1nase IPharmac1a 10 unitsiiJII

Method 1. Synthesize unlabelled rRNA by in v1tro transcription as descnbed 1n Volume I, Chapter 1, Protocol 3 2 . In a m1crocentrifuge tube mix: • rAN A substrate (step 1) ~250pmol • React 2 buffer e CIP • DEPC treated H 2 0

21.11 0.251.11 to 201.11

Contmued 146

Cathleen Enright and Barbara Sollner-\Vebb Protocol 4. Contmued 3 . Incubate the mixture at 37 °C for 20 min to dephosphorylate the 5' termmus of the rRNA. 4 . Inactivate the CIP by adding 51JI of 0 .1 M EDTA , 2.51JI of 20% SDS. 2111 of 10 mgl ml protemase K and H2 0 to 100 IJI, and heat the mixture at 65 °C for 30 min. 5 . Dilute the mixture to 250111 with 251JI of 3M sodium acetate, pH 5. 2, and 125111 of H2 0 6. Extract the rRNA w1th phenol:chloroform and ethanol prec1p1tate 1t (see Protocol 1 steps 6-9) . 7 . Label the rRNA by mixing the following m a microcentrifuge tube: • rRNA

volume containing 1 - 50 pmol of dephosphorylated RNA 5' ends

• 10 x kinase buffer [a-32P]ATP (150 1JCi)•

e e

31JI 15111 1 . 5 Ill

1 mg ml BSA

• T 4 polynucleotide kinase (20 units)

2 111

to 30 111 final volume • DEPC-treated H2 0 8 . Incubate the m1xture for 1 h at 37 °C. 9 . Isolate the labelled rRNA by electrophoresiS and ethanol prec1p1tat1on as described in Protocol 3 (steps 2-7) for internally-labelled rRNA. A longer exposure will be necessary to visualize end-labelled rRNA during autoradiography. • To generate unlabelled but 5' terminally-phosphorylated transcnpts, add 1 mM A TP 1n place of the radoolabelled ATP.

Protocol 5. In vitro preparation of 3' end-labelled rRNA substrate Equipment and reagents • Reagents for in v1tro synthesos of unlabelled rRNA substrate using T7 RNA polymerase (see Volume I, Chapter 1,

Protocol 31

e OMSO ISigmal • T4 RNA ligase IPharmacoa. 20 unitS I ~JII

• Oenatunng polyacrylamide (sequencongl gel and reagents for isolation of RNA as for onternally·labelled substrates (see above) e o.2 M OTT; store at -20 °C e 0.25M ATP

e o 1 mg.ml BSA

e ( 32P(pCp IICN, 3000CIImmol, 10,.,Cii~Jil • 10 x ligation buffer 10.5 M Tris-HCI. pH 7 .5, 0 .1 M MgCI 2 , 50 mM OTII, NB add OTT just before use from the 0 . 2 M stock solution

Contmued

147

5: Ribosomal RNA processing in vertebrates Protocol 5 . Continued Method 1. Synthesize unlabelled rRNA by in vitro transcription as described in Volume I, Chapter 1, Protocol 3. 2. In a microcentrifuge tube resuspend 20 pmol of the unlabelled rRNA in a mixture of 2,..1 of 0 .25 M ATP and 21JI of 0.1 mgfml BSA . 3. To the rRNA suspension add: e OMSO e [ 32P]pCp(1001JCi)•

21JI 101JI

• 10 x ligation buffer e T 4 RNA ligase 4 . Incubate the m1xture for

2 Ill 2 Ill ~

8 h at 16 °C.

5 . Isolate the labelled rRNA by electrophoresis and ethanol precipitation as described in Protoco/3 (steps 2-7) for internally-labelled rRNA. A longer exposure will be necessary to visualize end-labelled rRNA during autoradiography. • For the preparatiOn of unlabelled but 3 ' terminally-phosphorylated transcripts. add unlabelled pCp in place of the radiolabelled pCp (ref. 121

3.3 Preparation and use of mouse S-100 processing extract The mouse S-100 cell extract, whose preparation is described in Proroco/6, ~ commonly used for transcription of mouse rRNA genes (9) but also efficient!~ directs the processing of pre-rRNA at the 5 ' ETS site. The processing factor~ involved appear to be more evolutionarily conserved than are the RNA polymerase I transcription components, since the mouse S-100 extract also processes the 5' ETS site of human pre-rRNA (10). Internally labelled rRNA substrates (see Protocol 3) are generally used for this analysis. An efficien1 processing substrate need contain only the 200 nt conserved processing signal 3 to the actual cleavage site (31) of mouse pre-rRNA, but processing is more efficient when the sequence is extended by an additional 250 nt. The use of the S-100 extract for processing pre-rRNA is described in Protoco/7. The Dignam extract (32) also pro\ ides an efficient system in which to study the mouse 5' ETS processing. Protocol 6. Preparation of the mouse cell S - 1 00 processing extract8 Equipment and reagents • Mouse lymphoma cell line l1 21 0

1721JQ/ml streptomycin sulphate, 3.4 mM

e OMEM growth medium With 10% heatInactivated 156 °C for 30 min) horse serum , 3.02 g/1 sodium bicarbonate. 17 2 units ml penicillin G sodium ,

L-glutam~ne

• 0.2 M OTT; store at - 20 °C • Isotonic buffer (30 mM Hepes - KOH , pH 7.9, 0. 12 M NaCI. 1 mM OTTb)

Contmued

146

Cathleen Enright and Barbara Sollner-\\'ebb Protocol 6 . Continued • Low-salt buffer (10 mM Hepes-KOH, pH7. 9. 10mM KCI. 1.5mM MgCI 2 , 1 mM OTT". 0 . 1 mM EOTA ) • Oounce homogen1zer w ith type B pestle

• 5 x extract buffer (0 25M Hepes-KOH pH 7 9. 0 .75M KCI. 17 5mM MgCI2 , 5 mM on•, 0 .5 mM EOTA, 50% glycerol)

Method Carry out all steps at 0-4 °C

1. Grow mouse L1210 cells in DMEM growth medium to mid-log phase (5 x 105 cells mil in spinner flasks. 2 . Harvest the cells by pelleting them in plastic centrifuge bottles at 2000 g for 5 min . 3 . Wash the cell pellet twice w1th 10 vol. of isotonic buffer. 4 . Measure the packed cell volume. Resuspend the washed cells in 8 vol. of low-salt buffer and centrifuge them cit 2000 g for 5 min . 5 . Quickly discard t he supernatant. Resuspend the cells in 3 vol. las in step 41 of low-salt buffer and allow them to swell for 15 m1n at 0 °C. 6 . Homogenize the cells in the Dounce hom,)genizer us1ng 10 strokes of the B pestle. Ensure that virtually all the cells and a sizeable fraction of the nuclei have been broken by examining a drop of the lysate, spread on a glass slide and covered with a coverslip, under a microscope.

7 . Add 0. 25 vol. of 5 x extract buffer and centrifuge the mixture at 100 000 g for 60 min.

8 . Carefully remove the supernatant and divide it into 50-1 00 f.JI aliquots in microcentrifuge tubes. Freeze the tubes in .an ethanol/dry-ice bath and store them at -70 °C. • Based on ref. 9 . • Add the on just prior to use from the 0 .2 M On stock solution.

Protocol 7. Processing of pre-rRNA substrates in the mouse S-1 00 extract 8

Equipment and reagents strate (1 0 fmol 1n 2 ~I of H2 0) synthesized as in Protocol 3

• S-100 processing extract and 0.2 M OTT (see Protocol 61 • 5 >< S- 100 processmg buffer (50 mM Hepes-KOH, pH 7.9, 0.45 M KCI, 5 mM MgCI 2 , 3 mM on•. 0 .5 mM EOTA, 35% glycerol) • Internally-labelled

r

• 0 .1 M EDTA • 10mg.ml tRNA

• 10 mM ATP (neutralized to pH 7 .5) 32

• 3.0 M sodium acetate, pH 5.4

P I pre-rRNA sub-

• Phenol:chloroform 11.1 v/v)

Continued 149

5:

Ribosomal RNA processing in vertebrates

Protocol 7 . Contmued • Formamide 'dye mixture 195 % formamide . 5 mM EDTA. 0.03% xylene cyanol, 0 .03% bromophenol blue)

• 10 :< TBE electrophoresis buffer (50 mM Trts-borate pH 8 3. 1 mM EDTA I

• Denaturmg 4 % polyacrylamtde;9 M urea (sequenctngl gel (20 em long, 0 4 mm thtck, w idth dependent on sample number) in TBE buffer , and electrophoresis equipment See Volume I. Chapter 1, Protocol 8 or Volume I. Chapter 4 Protocol 11

Method 1 . In a mtcrocentrifuge tube on ice mix: 51-11

• 5 x S- 100 processing buffer

3 .751-11

e 10mM ATP •

[ 32 P] pre-rRNA

21-11

substrate ( 10 fmol)

51JI to 251-11

• S-100 processing extract e H2 0

2. Incubate the reaction for 25-45 min at 30 ° C. 3. Dilute the mixture to a final volume of 0 .2 ml wtth 20 IJI of 3M sodtum acetate, 201-11 of 0.1 M EDTA . 21-11 of 10 mg/ml tRNA (carrier) and H 2 0. 4 . Extract the sample with an equal volume of phenol :chloroform, ethanol precipitate the rRNA, wash the RNA pellet in 70% ethanol, and dry it as descnbed in Protocol 1, steps 6-1 0. 5 . Resuspend the rRNA in 5 1-11 of formamide/dye mixture. 6. Pre-run the sequencing gel for 10 min or until warm using TBE electrophoresis buffer. Rinse the wells of the gel with TBE buffer to remove urea. 7 . Heat the rRNA sample at 95 °C for 3 min and load it on to the pre-run (warm! sequencing gel. Run the gel until the required separation is achieved; this will depend on the sizes of the RNAs concerned. 8 . Vacuum dry the gel. There is no need to pretreat the gel prior to drying. 9 . Visualize the rRNA products by autoradiography (see Volume I, Chapter 1, Protocol 8 l • Adapted from refs 10, 31 , 33, 34. • Add the OTT just prior to use from the 0.2 M OTT stock solutton. < Detonize the formamtde as tn footnote b. Protocol 1.

l

' - - - - - - - - - -Figure 3A illustrates the use of the mouse S-100 processing exrract for tle clea\ age of the 5 ETS in mouse pre-rRNA.

3.4 Preparation and use of nucleolar and cytoplasmic processing extracts from HeLa cells Processing at the 5' border of the ISS region, which cannot be achieved using the mouse S-100 extract, occurs efficiently in an alternative two-stage procedure. 150

Cathleen Enright and Barbara Sollner-\Vebb

A

1 2 -

precursor

-

product

1 2

34

B

precursor - -

a

b~ c f

=/

-~ -\...product J b

·

Figure 3 In varo rRNA process1ng. !AI 5 ET5 processmg m the mouse S-1 00 extract. Up_ labelled pre rRNA conta1mng the mouse 5' ETS processing sequence IIane 11 was used as a substrate 1n the mouse 5 100 process1ng reaction !lane 21• the RNA precursor and product were resolved by electrophoresis and Visualized by autoradiography The procedure IS described in Protocol 7 Adapted from ref. 10. (8) Processing at the 5' end of the 185 region in Hela c:ell extracts. 32P·Iabelled pre-rRNA containmg the 5'-end ol the human 185 region (lane 1) was used as substrate 1n a processing reaction mvolving the Hela cell nucleolar extract !lane :21 Bands a. b. and c represent rRNA molecules cleaved. respectively, 8 nt before, 3 nt before. <>r 1 nt after the 5'·end of the 185 reg1on generated m v1vo. Fragment b was pur·fied by l!lectrophoresis IIane 3) and used as the substrate 10 a second processing reacuon mvolvmg the Hela cell P 100 cytoplasmiC extract (lane 4). Adapted from ref 12 w1th permission

T hus, this processing can be produced in l'itro with human pre-rRNA by first using a Hela cell nucleolar extract which main!> clea,es the pre-rRNA at a position either 3 or 8 nt upstream of the S ' -end of the ISS region ( 12). Then, using a P- 100 cytoplasmic extract, the 3 nt extension of the former intermediate can be trimmed off to generate the correct (in vi~·o) S -end of the 18S region (12). In addition, the nucleolar extract also catalyses the processing of the human .5' ETS. A nucleolar extract prepared in the same wa> from mouse cells appears to have similar processing activities to the HeLa cell nucleolar extract (T. Nilsen, persona l communication ; S. Kass and B. Sollner-Webb, unpublished data). The preparation of these two extracts and their use in the processing reaction are described in Protocols 8 and 9. Successful processmg requires a 300 nt synthetic pre-rRNA substrate containing the 18S rRNA processing site. 1 51

5: Ribosomal RNA processing in \'ertebrates Protocol 8. Preparation of nucleolar and P-1 00 cytoplasmic processing extracts from Hela cells8 Equipment and reagents • Uitracentnfuge, rotor. and tubes !Beckman Ti50 or equivalent, e V1rt1s 60 homogemzer (or equivalent) • Oounce homogemzers (w 1th a B type pestle and a Teflon pestle) • Hela cell suspension culture 145 x 105 cells ml) • Growth medium (SMEM; M . A. Bioproducts) supplemented w1th 7.5% horse serum • PBS 120 mM potass1um phosphate, pH 7.4. 0. 13 M NaCI) • 0.1 M PMSF (freshly made 1n ethanol) • o .2M on • 3M KCI • Dextran sulphate

• Solution A (10mM Tns-HCI, pH 7.5, 1 5 mM MgCI2 , 10 mM NaCI, 50 ll9 ml dextran sulphate) • Solution A ' contammg 0.5% Triton X-100 • Solution B (0.25M sucrose. 10mM MgCI 2 ) e Solut1on c 10.88M sucrose, 50mM MgCI 2 ) • Solution 0 l10mM Tns- HCI. pH 7.5, 50 mM MgCI2 • 10 mM NaCI, 50 IJQiml dextran sulphate) • Solution E (20 mM Tris - HCI, pH 7.5. 0.2mM EOTA. 0.5mM PMSF", 0 .5mM on<, 20% glycerol) • Protem assay k1t (Bio-Radl

Method NB All centrifugation steps are at 4 °C

A. Preparation of the HeLa cell nucleolar extract 1 . Grow the Hela cells in suspension culture in growth medium to a density of 4 x 1os to 6 x 1os cHIIs/ml. A total of 2 x 109 - 4 x 109 cells is required . 2 . Harvest the cells by centrifugation at 1000 g for 5 min. 3 . Measure the packed-cHI! volume and wash the cells with 100 vol. of PBS 4 . Resuspend the washed cells in 5 vol. of solution A and incubate the cell suspension for 10 min on ice. 5 . Homogenize the cells in the Dounce homogenizer, using the B pestle, until lysis is > 95% complete, as visualized under a microscope. 6 . Add an equal volume of solution A' (contaimng 0.5% Triton X·1 00) and collect the nuclei by centrifugation at 1500 g for 5 min. 7 . Carefully remove the supernatant and save it for the immediate preparation of the P-1 00 cytoplasmic extract (steps 19-23). B. Resuspend the nuclei in 10 vol. (referring to the original cell volume. see step 3) of solution B and layer this over a cushion of an equal volume of solution C in a Beckman T i50 centrifuge tube. 9 . Centrifuge the gradiemt at 2500 g for 10 min. 10. Resuspend the purified nuclei at 2 x 107 nuclei/ml in solution D (count nuclei in a haemocytometer). 11 . Lyse the nuclei by shearing the solution tor 11 min at 5000 r.p .m . in a V1rtis 60 homogeniz•ar fitted with the macroblade.

Continued

152

Cathleen Enright and Barbaro SolJner-Webb Protocol 8 . Contmued 12. Layer th1s suspension of lysed nuclei over an equal volume of solution C and centrifuge the gradient at 3500 g for 15 min to recover the nucleoli. 13. Resuspend the punfied nucleolar pellet in an equal volume of solution E. Add 3M KCI to a final concentration of 0 .5 M . 14. Incubate the nucleoli on 1ce for 10 min w1th gentle agitation. 15. Dilute the nucleolar extract with solution E so that the final concentration of KCI is 0.12 M . 16. Clarify the extract by centrifugation for 10 min at 225 000 gin a Beckman Ti50 rotor. 17. Carefully recover the supernatant. Determine its protein concentration using the Bio-Rad protein assay kitd. 18. Store the extract in aliquots (containing, - 0 . 5 mg of protein per mil at - 70 ° C .

B. Preparation of the HeLa cell P-100 cytoplasmic extract 19. Clarify the supernatant from step 7 by centrifugation at 20 000 g for 10 mm. 20. Carefully recover the supernatant and centrifuge it at 100 000 g for 60 m1n 1n a Beckman Ti50 rotor. 21 . Discard the supernatant and quickly rinse the surface of the pellet with solut1on E. 22. Resuspend the pellet in 0 .2 vol. (calculated from the original cell volume as in step 3) of solution E by gentle homogenization in a 1 ml Dounce homogenizer fitted with a Teflon pesth~. 23. Store the resulting extract in small aliquot s at - 70 °C. Typically the protein concentration of the P- 100 extract is - 10 mg/ml. • Based on ref. 1 2. b Add PMSF just before use from the 0 . 1 M stock solution. c Add on JUSt before use from the 0.2 M stock solution. d See also ref. 35.

Protocol 9 . Use of Hela cell nucleolar and P-100 cytoplasmic extracts for processing of the 5' terminus of human 1 8S rRNA" Equipment and reagents •

32 P-Iabelled pre-rRNA substrate extend· ing from - 70 to + 165 (corresponding to restriction Sites Ncrl and X ball relative to the 5'-end of 185 rRNA , both 5 ' and 3 ' end-labelled RNAs are required (see Protocols 4, 5) .

• Hela

cell

nucl eolar

and

P 100

cytcoplasm1c extracts and solution E as in P' otocol 8 . • 5 x nucleolar process1ng buffer (0 32 M KCI, 40mM Tris-HCI, pH 7 .5, 10mM MgCI2 • 1 mM sperm1dinel e OEPC-treated H 2 0, gel elution buffer and glycogen carrier as in Protocol 3

Contmued

153

5: Ribosomal RNA processing in vertebrates Prot o col 9 . Continued • Sequencing gels. electrophoresis equ•pment, formam•de dye maxture. TBE buHer as in Protocol 7

• Phenol:chl oroform , 3.0 M sodaum acetate. pH 5.4, 0 . 1 M EDTA, 10 mg ml tRNA as i n Protocol 7

Method A. For the imtial cleavage

Set up a cleavage reaction for each of the end-labelled pre-rRNA substrates (5'- or 3'-labelledl 1. Max the following an a microcentnfuge tube on ice: • 5' or 3 ' end-labelled pre-rRNA substrate

volume contaanang up to 500 ng

• 5 x nucleolar processing buffer

41-JI

• Hela cell nucleolar extract

21-JI

e DEPC-treated H 2 0

to 20 IJI final volume

2. Incubate the samples for 2-3 h at 30 °C. 3. Recover RNA products as in Protocol 7 steps 3-4. 4 . Analyse RNA products on 40 em long, 6% acrylam1de. 7 M urea sequencing gel as in Protocol 7, steps 5-9 B

Final trimming of the rRNA intermediate with the 3 nt extenston 5. Isolate the appropriate s1zed 3' end-labelled RNA intermediate (i.e. the RNA wtth the 3 nt 5 extenston) after the gel electrophoresis (step 4 ) as descnbed an Protocol 3. step 3 6. Recover the rRNA antermediate by ethanol precapitation !Protocol 1, steps 7-9}. 7 . Dilute the P-1 00 cytoplasmic extract with 9 vol of solution E 8 . Set up the second stage processing reaction as tn step 1 by mixing: • isolated rRNA intermediate (step 6)

11 1-11

• diluted P-1 00 extract (step 7)

5~-JI

• 5 x nucleolar processtng buffer

4 1-11

9 . Incubate the mixture at 37 °C for - 1 h. 101 Dilute the maxture to 1001-11 with 101-11 of 3M sodium acetate, pH SA 101-11 of 0.1 M EDTA, 20 IJg glycogen carrier, and H 2 0 . 11 . Phenol:chloroform extract and precipitate the RNA

6-9 Based on ref . 12.

154

~san Protocol

7, steps

Cathleen Enright and Barbaro Sollner-Webb T ypical data obtained using the Hela cell nucleolar and P-100 cytoplasmic are illustrated in Figure 38. RNase Tl fingerprint analysis (described in ref. 12) can be employed to verify the precision of the processing reactions by examining the termini generated (see also Volume I, Chapter 5, Section 8). e:~tracts

3 .5 Cleavage of pre-rRNA using purified nucleolar

endonuclease An endonuclease which can be isolated from mouse nucleoli has been shown to deave mouse pre-rRNA at a limited number of positions (approximately one cleavage every 50-75 nt). One of these preferential cleavage sites is identical to the position of the 5' ETS processing site used in vivo. The relevant procedures are described in ref. II . The relationship of this cleavage reaction to the 5' ETS processing which takes place in the various extracts described in Sections 3.3 and 3.4 remains to be determined. It does not involve the U3 snRNP. It may be that there are alternative means of achieving this processing, or that the processing in extracts may involve this endonuclease '"ith additional factors being required ro achieve site specificity. Alternatively, the cleavages by the endonuclease (which is single-strand specific) occur at the single-stranded loops which are predicted to occur in the secondary structure of the pre-rRNA.

3.6 Use of deletion analysis in the investigation of rRNA processing To determine the nucleotide sequences (or structural domains) of the pre-rRNA thatt are necessary to direct the in vitro processing reactions, a systematic series of altered rRNA substrates can be transcribed from appropriately constructed muttant rONA templates. These can be assayed for processing efficiency by the methods described in Sections 3.3 and 3.4. Procedures for preparing mutant templates containing 5 1 or 3' deletions, block substitution mutations, or point mutations, will be found in standard molecular biology/ laboratory manuals (e.g. ref. 23). lllustrations of the use of such mutant substrates to analyse the sequence requirements for the 5 1 ETS processing of mouse pre-rRNA are to be found in refs 31 and 33.

4. Analysis of protein-RNA associations 1n rRNA processing Since all the experimental evidence available indicates that metazoan rRNA processing events are not autocatalytic, there is considerable interest in learning about the cellular components that are responsible for carrying out the processing reatctions. A number of methods are available for exploring the association of proteins with pre-rRNA substrates. These are described in the following sections as applied to the 5' ETS processing in mouse. 155

5: Ribosomal RNA processing in vertebrates

Protocol 10 . Gel-shift assay for analysis of rRNA processing complexes8 Equipment and reagents

•s- 100

processmg extract, 5 x S-100 processing buffer, 32P-Iabelled pre-rRNA substrate, 10 mM A TP. TBE buffer, and formamide.dye mixture (see Protocol 71

• 20 mg rml heparin (Sigma) in DEPCtreated H 2 0 (see Protocol 3) • Non-denaturing 4% polyacrylamide gel (2.0-2 5 mm thick; 25 em long;

acrylamide: bisacrylamide, 65:1 l n 0. 5 x TBE buffer. electrophoresis equipment (preparation of non-denaturing polyacrylamide gels follows that of denaturing gels described in Volume I Chapter 1, Protocol 8. or Volume I Chapter 4, Protocol 11, except that the urea Is omitted)

Method 1. Set up the processing reaction as described in Protocol 7, step 1. 2. Incubate the mixture for 25-45 min at 30 °C, and then place the tubes on ice. 3 . Dilute the stock 20 mg/ml heparin solution to 0 .35 mg/ml with H 2 0 Gently add 21-11 of 0.35 mg/ml heparin to the incubation m1xture (step 2 to remove loosely assoc1ated protems. Do not m1x the contents of the microcentrifuge tube after the heparin addition. 4. Incubate the reaction for an additional 10 min at 30 °C and return it to ice. 5 . During the processing reaction pre-run the non-denaturing polyacryl amide gel at 13V/cm for 1 h using 0.5xTBE buffer as the electrode buffer. 6. Load 1 2. 5-1 51JI of the reaction mixture onto the pre-run gel. Load a equivalent amount of 32P-Iabelled pre-rRNA substrate that has not bee exposed to S- 100 processing extract in an adjacent well. Also load a sample of formamide/dye mixture into an empty well. 7. Electrophorese the samples at no more than 1 3 V /em (the gel should never be warm to the touch) until the xylene cyanol dye has travelled the length of the gel. 8 . Dry the gel onto Whatman chromatography paper and visualize the pre rRNA substrate and the rRNA processing complex by autoradiography

• Based on ref. 34.

156

Cathleen Enright and Barbara Sollner-Webb

4.1 Gel-shift analysis Factors present in the mouse S-100 processing extract (see Protocol 6) that catalyse the 5' ETS processing can be identified by their ability to form a specific complex with the pre-rRNA substrates, but not with processing-incompetent RNA which does not possess the necessary processing signals. The complex 1s detected by the electrophoretic retardation of the RNA substrate (gel shift or mobility retardation). The gel-shift assay is described in Protocol 10 using a radiolabelled RNA substrate and visualizing the complexes directly by autoradiography. The results of a typical gel-shift assay are shown in Figure 4. The specificity of the gel-shift was demonstrated by competition analysis (Figure 48). The extract was pre-incubated with an unlabelled RNA-either a processingcompetent rRNA (sp) containing residues 645·-668 of the rRNA transcription unit which comprise the 5 ' ETS processing site or a processing-incompetent rRNA (nsp) devoid of these nucleotides (Figure 4A ). The former, but not the latter, competitor rRNA competes with the labelled RNA substrate added subsequently for the assembly of 5' ETS ribonucleoprotein processing complex components. The processing complexes formed on pre-rRNA substrates can also be visualized by carrying out Northern blotting analysis after the electrophoresis, probing the blotted complexes with 32P-labelled nucleic acid probes complementary to specific rRNA sequences. In this case, perform the processing reaction (Protocol 10, step I) with 80 fmol of 3 H-labelled rRNA synthesized as described in Protoco/3. Volume I, Chapter 2 (Section 3.6), Volume I, Chapter 4 (Section 7 .5), and ref. 23 describe protocols for Northern analysis. Negligible amounts of endogenous rRNA from the mouse S-100 processing extract appear in the gel and are transferred to the Northern !blot, so background signals due to endogenous rRNA are minimal. Figure 5 illustrates the use of Northern blot analysis following the gel-shift assay. Here the presence of smaJI nuclear RNAs was shown using an unlabelled rRNA substrate and a radiolabelled hybridization probe for the snRNA. One example is the U3 snRNP which is present in complexes formed on the rRNA that extends from the 5' ETS processing site to residue - 1060 of the mouse rRNA transcription unit. Because the U3 snRNA is non-specificalJy associated with extract proteins fractionated throughout the gel lane, probing the complexes formed on different sized rRNAs, which have distinctive mobilities, permits assurance that the snRNA is indeed specifically associated with the input rRNA.

4.2 Analysis of rRNA associations by rate zonal centrifugation The 5' ETS processing complex can also be resolved by rate zonal centrifugation as described in Protocol 11. 157

5: Ribosomal RNA processing in vertebrates

A

t r:t~

18S

5as

28S

850

"l

I

645 _ ,_ _ _ _ _ __ sp

669--------

B

2

3

4

nsp

c

2

3

250 -

110 - 958575-

spec If lc complexes

ca~~pet I tor -

• +

extract competitor

hot RNA

+

sp nsp

+

sp nsp

+

+

+

+

Figure 4 . Gel-shift and UV cross-linking analys1s of the mouse 5 ETS processing complex. IAl The mouse 5' ETS process1ng reg1on . The top of the diagram shows the 5' ETS processmg site 1n the mouse rRNA transcription umt. The 200 nt process1ng s1gnal (residues - 650-8501 1s shown expanded 1n the middle of the diagram Below are shown the spec1f1c lsp) and non· spec1f1c (nspl competitor rRNAs used 1n parts 8 and C. wh1ch start at rRNA residues 645 and 669, respectively. (8) Gel-shift analysis. 32P-Iabelled mouse pre·rRNA containing the 5' ETS processing signal (residues 546 - 875) was incubated w1th the mouse S-1 00 extract and

158

Cathleen Enright and Barbara Sollner-Webb Protocol 11. Analysis of the 5' ETS processing complex by rate zonal centrifugation"

Eqwpment and reagents • S- 100 processtng extract. 10 mM ATP 5 xS-100 processmg buffer, and 3 'PIabelled pre-rRNA substrate as 1n

Protocol 7 • S ·100 processing buffer lackmg glycerol plus the same buffer containing 10% glycerol and cont81t1ing 30% glycerol

• Gradient former (e.g. Hoefer) and dens1ty grad1ent fractionator le g. ISCOI • Ultracentrifuge tubes, and rotor (e g. Beckman SW55 or equivalent) • 20 mglml he paron

Method I . Set up a processing reaction as described 1n Protocol 7 (step 1) but scaleup the volume of the react1on to 751JI :~.

Incubate the reaction for 50 mtn at 30 °C

:~ .

Place the react1ons on ice. add heparin, and continue the incubation as described in Protocol 10 (steps 3 - 4).

4. Dilute the processing m1xture with an equal vol. of S-1 00 processing buffer lacking glycerol. ~i .

Set up a 5 ml, 10-30% glycerol gradient using S-100 processing buffer contatntng 10% glycerol and S-1 00 processing buffer containing 30% glycerol using the gradient former according to the manufacturer's instructions.

Ei. Load the processing reaction (step 4) onto the gradient and centrifuge it at 40 000 r.p.m in a SW55 rotor for 5 h at 19 °C.

Continued

the resultant nucleoprotein complexes (speCific complexes) were resolved electrophoret1cally IIane' 2). Lane 1 contams the pre·rRNA substrate alone Lanes 3 and 4 demonstrate the spec1fic1ty of the gel-sh1ft As indicated in the table below the autoradiograph. the extract in lane 3 was ftrst pre-incubated With a 12-fold molar excess of unlabelled spec1fic competitor rRNA lsp; processing-competent rRNA extending from res1dues 645-875: see panel A ) In lane 4 , unlabelled non-spec1fic competitor was used (nsp: processmg-1ncompetent rRNA extendtng from res1dues 669-875; see panel A) Both extracts were then tncubated With 32 P-Iabelled pre-rRNA (hot RNA). (C) UV cross-linking The polypeptides labelled when 32 P-Iubelled, 4 -S -UMP substituted pre-rRNA was mcubated w1th the mouseS 100 processmg extrc:1ct. subJeCted to UV cross-link1ng and liberated after RNase d1gest1on are shown 1n lane 1. As Indicated at the bottom of the autoradiograph, the specifiCity of the UV cross·ltnk1ng was JUdged by the effects of pre-1ncubat1ng the extract w1th either the spec1ftc (sp) or nonspecific (nsp) competitoor rRNA (see panel AI. Polypeptides on the s1ze range 75-250 kDa were eliminated by pre-incubation w1th the spec1f1c rRNA IIane 21 but not with the non-spec1f1c rRN.a, (lane 31. Indicating that they are specifically assoc1ated with the rRNA . In contrast, th1s pre-111cubation had no effect on polypeptides smaller than 75 kDa. (Adapted from ref 34.)

159

5: Ribosomal RNA processing in vertebrates Protocol 11 . Continued 7. Fractionate the gradient using the gradient fractionator. Collect 250 IJI fractions. 8. Count an aliquot of each fraction in a scintillation counter to determine the distribution of radioactivity across the gradient. 9 . Use the remainder of each fraction for subsequent analysis by gel-shift assay (see Protocol 10) or for isolation of rRNA use one of the methods described in Volume I, Chapter 1, Section 3. • From ref. 34.

4.3 Use ofUV cross-linking to detect polypeptides associated

with rRNA in processing complexes To label the polypeptides that are specifically associated with the rRNA 5' ETS processing signal, UV cross-linking has been employed (34). The experimental procedure involves the synthesis of pre-rRNA substrate substituted with a rhionucleotide, 4-S-UMP. The precursor for the in vitro transcription of 1his

•

Figure 5 . Analysis of U3 RNA 1n electrophoretlca11y separated complexes. Processing react.ons containing the indicated RNAs (rRNA substrates (312-1290 and 645-1 2901 and he processing-mcompetent RNAs (rRNA 669-1290) and a 900 nt pGEM transcript ) were incubated as in Protocol 7, electrophoret1callv resolved on non-denaturing gels as in Proco.:ol 10, blotted , and analysed with a probe for U3 sequences. The solid arrow represents the positions of specific shifted complexes.

160

Cathleen Enright and Barbara Sollner-Webb substrate, 4-S-UTP, is prepared by phosphorylation of 4-S-UDP as described in ProfOco/12. The substituted rRNA substrate is then used to form a processing complex \\-;th a mouse S-100 processing extract (see Proroco/13). The processing complex is then irradiated with UV light to cross-link regions of the rRNA substrate to their associated proteins in the complex. The rest of the rRNA is then removed by digestion with RNase A and RNase TJ. Finally, SDS-PAGE is used to separate the cross-linked protein:RNA complex. Protocol 12 . Synthesis of 4-S-UTPa Reagents • 1 .0 M Tris-HCI. pH 7.6

• 0 . 2 M phosphoenolpyruvate (PEP)

• 30M KCI

• 20mM NADH

• 1.0 M MgCI 2 • 0. 2 M and 0 .1 M OTT stock solutiOns

• Lactate dehydrogenase (LDH)

• 17 mM 4-thlo-UDP

• Pyruvate ki nase (PK )

• 01MATP

• Nucleoside diphosphate kinase (NDKI

Method 1. Make up reaction buffer by mtxing: e 1.0 M Tris- HCI, pH 7.6 400 )JI e 3.0 M KCI 53 )JI e 1.0 M MgCI2 e 0 .2 M OTT e H2 0

48)JI 5 )JI 3.494ml

2. Make PK/LDH mix by dissolvtng PK and LDH in reactton buffer ( - 0 . 7 5 U )JI PK and -1 .0U.)JI LDHI. 3. In a mtcrocentnfuge tube mtx the followtng e 1.0M Tns-HCI, pH 7. 6 100)JI e 3.0M KCI e o 1M OTT e 1.0 M MgCI2 e 1 7 mM 4 -thio-UDP e Q. 1 M ATP e 0.2M PEP e 20mM NADH e PK LDH mix e 4 )JI NDK • H 20

13.31JI 2 .5)JI 12 )JI 100 )JI 30)JI 40)JI 64)JI 4 . 3)JI 4)J) to 1.0 ml f inal volume

Continued 161

5. Ribosomal RNA processing in vertebrates Prot ocol 12. Continued 4 . Incubate the mixture at 37 °C for 1 h. keeping as light-free as possible. 5

Extract the mixture With phenol and then With ether Store the solution at - 70 °C, in foil-covered tubes. Under these conditions about 70% of the 4-thio ·UDP is phosphorylated to 4 -thlo-UTP.

• Adapted from ref. 36.

- ., Protocol 13 . Analysis of the proteins associated with processing complexes using UV cross-linkinga Equipment and reagents • 5 x transcnpt1on buffer (0. 2 M TnsHCI, pH7.5, 30mM MgCI,. 10mM spermidine) • ATP GTP soluuon 15 mM each) neutralIZed to pH 7 5: store at 20 °C • Separate 5 mM A TP and 10 mM CTP solutions. neutralized to pH 7 5, store -20 °C e (a-3 'P ] CTP UCN; 3000 CtJ mmol, 101JCi 'l·lll

• Gel elution buffer and glycogen as 1n Protoco/3 • S 100 processing extract lsee Protocol

6 • 5 x 5 · 1 00 process1ng buffer I see Protocol 7) With NaCI replac1ng the~ (' • Extract buffer (50 mM Hepes-KOH pH 7 .9, 0.15 M NaCI, 3. 5 mM MgCI 1 mM DTTI • 20 11g ·ml hepann • Trans1ilum,nator 1>. = 300 nm. 7 mW,cm1 : FotoPrep 1 from Fotodyne or equivalent I

• 2mM 4-5 -UTP (see Protocol 12)

e o.2 M OTT • Uneanzed template DNA (plasm1d con· tain1ng the appropnate rRNA sequence cloned ad1acent to the T7 phage promoter; 0 . 25 mg.ml) e T7 RNA polymerase IBRL; 50 U/111) • RNasm !Pro mega) • Oenatunng polyacrylamide (sequencing) gel, formam1de dye mixture . TBE buffer as in Protocol 7

• RNese A IBRL 10 mg.mil e RNase T 1 IBRL 200 U 'IJI) • 3 x SOS-gelloading buffer 10 18M Trrs HCI. pH 6 .8. 1 5 M 2 -mercaptoethar>ol, 30% glycerol. 6% SOS, 0. 1% bromo phenol blue) • SDS- PAGE gel 110% acrylam•de prepared accord1ng to Volume I. Chapter 5. Protocol 7 or ref 371

Method Preparation of the pre-rRNA substrate substituted with 4 -S-UMP

1. Synthesize the pre-rRNA substrate substituted w1th 4 -S UMP by setting up the following reaction (in the order given) in a microcentrifuge tube on ice:

• H2 0

volume to achieve 48 5 Ill final volume

• 5 x transcription buffer e o 2M OTT

10~JI 2~JI

Contmued

162

Cathleen Enright and Barbara Sol/ner-\Vebb Protocol 13. Continued • RNastn

21JI

e 5 mM A TP GTP solutiOn

51JI 4.51JI

e 10mM CTP e 2 mM 4 -S-UTP e [a-32P] CTP (120 llCil

51JI 121JI

• template DNA (0. 7 IJgl

31JI

2. Add 1. 51JI of T7 RNA polymerase and mcubate the reaction at 37 ° C for 90 mm, keeping 1t as light-free as poss1ble. 3 . Isolate the 32P-Iabelled, substituted rRNA product by electrophoresis on a sequencing gel, as described in Protocol 3, steps 2-7 4. Calculate the yield of rRNA product as described m Protocol 3, steps 8-12, using UTP as the marker nucleotide. Preparation of processmg complexes using 4-S-UMP substituted pre-rRNA substrate

NB Protect the process1ng react1ons from light m steps 5-7 to mm1m1ze spurious cross-linking. 5 . Set up a 50 IJI processing reaction, as described in Protocol 5, (step 1) by mixing : • 4 -S-UMP substituted rRNA (step 3) • S· 100 processing extract e 5mM ATP

volume contaming 10 fmol 31JI

• extract buffer • 5 x S· 100 processing buffer (With NaCI in place of KCI)

10111

• H2 0

to 50 IJI fmal volume

6 . Incubate the reaction for 50 min at 30 °C and then place 1t on ice 7 . Add 2 Ill of 20 IJg/ml heparin and incubate the mixture for an additional 10mm at 30 ° C. Cross-/inkmg of processmg protems to rRNA and SDS-PAGE analysis 8 . Transfer the sample to one of the wells 1n a 96-well m1crot1tre plate and place the plate 1n a Pyrex dish contaming just enough cold water to allow the microtitre dish to float. 9 . Place the Pyrex d1sh on top of the UV transilluminator and irradiate the sample for 1 5 m1n using the analytical power settmg 10. In a m1crocentrifuge tube, m1x 40 IJg of RNase A and 200 units of RNase T1 tn H 2 0 to a final volume of 20 IJI. 11 . Transfer 20 Ill of the irradiated processing sample (step 9) to the microcentnfuge tube containing the RNa se A and RNase T1 (step 10) and incubate the mixture for 1 5 min at 30 ° C

Continued 163

5: Ribosomal RNA processing in vertebrates

Protocol 13. Contmued 12. Add 20 111 of 3 x SDS-gelloading buffer to the digested sample and heat the m1xture for 3 mm at 95 ° C .

13. Load the sample onto the SDS-PAGE gel and separate the protems as descnbed 1n Volume I. Chapter 5, Protocol 7, or ref . 37 14. ldent1fy the proteins cross-linked to the nuclease-resistant 32P-Iabelled RNA residues by autoradiography. • Ba:sed on ref. 34.

Representative data from a UV cross-linking experiment are shown in Figure 4C. Polypeptides that become labelled when using a radiolabelled processingcompetent rRNA substrate, but not when using a radiolabelled processingincompetent rRNA substrate, are deemed to be specifically associated. Pre-incubation of the processing extract with unlabelled processing-competent or processing-incompetent rRNA substrates before the addition of the labelled 4-S-UMP substituted substrate affords confirmation of specificity. The subsequent labelling of specifically associated polypeptides will be aboli 11ed by the former but not by the latter competitor RNA as shown in Figure 4C.

5 . Assessment of the requirement for cellular RNA in

rRNA processing Section 4 described methods whereby processing polypeptides associated with pre-rRNA could be identified. Processing of pre-rRNA may involve the participation of several cellular RNAs, e.g. U3 snRNA. This section describes micrococcal nuclease treatment and oligonucleotide-directed RNase H digestion of RNAs in a processing extract which can be used to provide evidence for the requirement of cellular RNA in the processing reaction.

5.1 lMicrococcal nuclease digestion If treatment of a processing extract with micrococcal nuclease (but not a 'mock' treatment) destroys the processing acthrity, and this is not restored b) the addition of non-specific RNA, it can be presumed that the processing reaction has a requirement for one or more specific nucleic acids. Lack of sensu '>'ity to DNase I can further suggest that the nucleic acid is RNA. Note that an ab~e:1ce of an effect of micrococcal nuclease is not proof that an RNA is not reqw ed; a required RNA may not be accessible to the nuclease. Protocol 14 describes the treatment of the mouse S-100 processing extract with micrococcal nuclease. The advantage of micrococcal nuclease for this type of analysis is that it is a Ca2 + -dependent nuclease. This allows it to be inactivated once digestion of a putative nucleic acid is complete, using an excess of the Ca 2 + -chelator EGTA, so that the processing activity of the treated extract can then be tested. 164

Cathleen Enright and Barbara Sollner-\Vebb The length of time required for micrococcal nuclease digestion of the extract should be determined in a pilot experiment, but the time in Protocol 14 has been used successfully in the author's laboratory.

Protocol 14. Treatment of the mouse S-1 00 processing extract with micrococcal nuclease8

Equipment and reagents • S-1 00 process1ng extract (see Protoco/6) e 10mM CaCI2 • Micrococcal nuclease (BRL, 1 mg ml) e 60mM EGTA , pHS.O • Equipment and reagents for studymg

processing of pre·rRNA as described m Protocol 7

• Modified 5 x S 100 processmg butler (0.42 M KCI, 40 mM Hepes-KOH, pH 7.9, 16 mM MgCI2 , 33% glycerol 2.6 mM DTI. 0.46 mM EDTAI

Method 1. Mix 6 )JI of the S- 100 processing extract with 1 )JI of 1 0 mM CaCI 2 and incubate the mixture for 30 sec at 30 °C. Cool the reaction mixture on ice. 2 . Add 2 )JI of 1 mg/ml micrococcal nuclease and incubate the extract for 90 sec at 30 °C . Cool the tube on ice. 3 . Add 1 )JI of 60 mM EGTA and incubate for 30 sec at 30 °C. Again cool the extract on ice.

4 . Test the processing activity of the treated extract as described in Protocol 7, except replace the 5 1.11 of S- 100 processing extract with the 10 )JI of the nuclease-treated extract (step 3 above) and substitute modified 5 x S-1 00 processing buffer. • From ref. 34.

Data from an experiment in which the mouse S-100 extract was treated with micrococcal nuclease and then tested for its ability to process pre-rRNA are shown in Figure 6A.

5.2 Oligonucleotide-directed RNase H digestion of cellular RNAs involved in pre-rRNA processing Oligonucleotide-directed RNase H treatment can be employed to determine whether a particular RNA is required for a processing event. RNase H digests RNA only in an RNA:DNA hybrid. Therefore, hybridization of an oligodeoxyribonucleotide complementary to a specific RNA thought to be involved in processing could render that RNA susceptible to RNase H digestion. If this RNA is digested by RNase H (checked by Northern blot analysis) and is an essential component of the processing extract, the extract will then be unable 165

5: Ribosomal RNA processing in vertebrates

A

8

--

pl"oduct

' I

..

""c "

-

C. C.,E C.

-

...

...

•I

'f•12

UJ o

':!

....

0

<0

"' "' "'... ...

~

-- 0

:>

2

~"----~

.... "'

OS

3

0

5

Figure 6 . Sens1t1v1ty of the mouse 5' ETS processmg reacuon to micrococcal nuclease or RNase H treatment of the mouse S- 100 processmg extract !AI Micrococcal nuclease sens1t1V1ty Samples of the mouse S- 100 processmg extract were treated as Indicated 1n tlle table below the autoradiograph. At the start It= 0), samples received CaCI2 (Ca; lanes 3 and 5) or CaCI2 and excess EGTA ICa.E; lane 4), or no add1t1on (lane 21 After 4 m•n It= 4Lmicrococcal nuclease IMN) was added (lanes 4 and 5). After another 4 m•n It =8), excess EGTA was added to all samples lackmg this reagent. Finally after a further 4 m1n It= 121 np. labelled pre-rRNA substrate was added to all the samples to test their processing ability The pre-rRNA (precursor) and processed product (product) were separated electrophoret•calty. lane 1 shows the untreated pre-rRNA. (Adapted from ref 381 IBI Oligonucleotide-directed RNase H sens1t1vity The S-100 extract was pre-incubated with RNAse H m the presence of no oligonucleotide (-oligo; lane 2), an oligonucleotide complementary to U2 snRNA (U2 L 15; lane 31. an oligonucleotide complementary to res1dues 64-79 of U3 snRNA (64-79; lane 4), or an oligonucleotide complementary to residues 80-96 of U3 (80-96; lane 5). All samples were then treated with DNase I followed by incubation with radiolabelled pre-rRNA processing substrate. Only the oligonucleotide targeted on residues 64- 79 of U3 snRNA resulted in RNase H digestion and loss of processing activity as shown by the absence of the (smaller) rANA product. Ne1ther the non-specific U2 oligonucleotide nor the U3 oligonucleotide targeted to the double-stranded reg1on of U3 snRNA (residues 80- 961 supported RNase H digest•on. (Adapted from ref 38).

to process the pre-rRNA. Oligonucleotides complementary 10 prominent singlestranded regions of the RNA are the most likely 10 be successful in directing digestion of this RNA in the presence of cellular components. For instance. of eight anti-U3 oligonucleotides tested, only one, corresponding to residues 64-79 of mouse U3 RNA, has been successful in promotmg RNase H directed remo\'al of U3 RNA activity from the mouse S-1 00 extract (38).

5.2.1 RNase H digestion of cellular RNAs in processing extracts The method is described m Protoco/15 and in Volume I, Chapter 4 (Section 6). Following RNase H treatment, assay an aliquot of the extract by Northern blot analysis to monitor the RNA digestion and test another ahquot for altered processing efficiency after incubation \\;th a 32 P-Iabelled pre-rRNA substrate. It is important to demonstrate that the loss of processing acti\ity occurs following 166

Cathleen Enright and Barbaro Sollner-Webb digestion of the RNA of interest, but not following parallel treatments with control oligonucleotides. It should al~o be remembered that an oligonucleotide may fortuitously allow destruction of an unanticipated RNA and that an apparent!~ negari' e result is hard to interpret unless one can be certain that the RNA destruction \\85 sufficiently complete to assure detection of an) potenual effect on processing.

Protocol 15. Oligonucleotide-directed RNase H digestion of RNA in the mouse S-1 00 processing extract" EqUipment and reagents • S · 100 processing extract and 0. 2 M D n stock solution, Protocol 6

e 5 x RNase H buffer 10.5 M KCI , 40% glycerol, 9 .76 mM DTI') • RNase free DNase IWorthmgton;

• Oligodeoxynbonucleotlde complementary to the target RNA sequence (1 1Jgi 1JI) • RNase H IBRL; 2 U/1JI)

2-5U'IIH • Equipment and reagents for studying processmg of pre·rRNA as described in Protocol 7

Method 1. Set up the followmg reaction mixture in a microcentrifuge tube on ice: • S-1 00 processing extract

10

~o~l

• oligonucleotide • 5 x ANase H buffer • RNase H

2 . Incubate the m1xture for 10 m1n at 37 °C, then for 20 min at 30 °C and then place it on ice. 3. Add 1 IJI of RNase -free DNase and incubate the mixture for 3 min at 37 °C followed by 7 min at 30 °C. 4 . Remove 6.4 IJI for Northern blot analysis of the targeted snRNA (see Volume I, Chapters 2 (Section 3.6) and 4 (Section 7 5)). 5 . Use the remainder of the reactiOn maxture from step 3 (9.6 jJI) to test the processing activity of the extract as descnbed 1n Protocol 7. For the processing assay, dilute the remainder of the reaction to 1 51JI with 10 fmol 32 P ·labelled pre-rRNA and A TP to yield a free magnesium 10n concentration of -0.5 mM. Although processing does not require ATP, including ATP 1n the reaction as opposed to adjusting the MgCI 2 to 0 5 mM results in sharper gel bands From ref. 38. Add DTI just before use from the OTT stock soluuon.

167

5: Ribosomal RNA processing in vertebrates Figure 68 shows the results of an experiment in which RNase H was used to demonstrate that U3 snRNA is required for 5' ETS processing in the mouse S-100 extract (38).

5.2.2 Oligonucleotide-directed RNase H digestion of RNAs in micro-injected oocytes The e111dogenous RNase H present in Xenopus laevis oocyres has been exploited to demonstrate the involvement of U3 snRNA on the processing of the cellular pre-rl~NA (16). In this case, the U3-specific oligonucleotide was injected tmo the oocyte nucleus and the endogenous RNase H performed the select e digest ion. It was concluded that the processing which occurs at the 5 ' -end of the 5 .8S rRNA region is dependent on the presence of U3 in the oocyte. Pr<:paration of the oocytes and micro-injection of material into the ooc te nucleus are specialized techniques\\ hich cannot be covered in the necessary de 4il in this chapter. For detailed instructions , refer to ref. 16. Protoco/16 describes the s111bsequent incubation and processing of the oocytes.

Protocol 16. Analysis of nucleic acid components necessary for rRNA processing using oocytes micro-injected with oligonucleotides8

Equipment and reagents • Oligodeoxynbonucleotide ( 1-2 mg/ml) complementary to the target snRNA • M od tl ied Barth's buller (88 mM NaCI, 1 mM KCI. 0. 82 mM MgSO, . 0 .33 mM calcium mtrate, 0 .41 mM CaCI2 • 10 mM Tris-HCI. pH 7.5, 20 mg/1penicilltn and 20 mg 'I streptomyctn l e [ u-32P ] UTP UCN; 800 Ci/mmol, 101JCi/1JI)

• Extraction buffer (0.2 M Tros-HCI , pH 7.5, 0 6 M NaCI, 20mM EDTA 2% SDS, 0 . 2 mgtml protemase Kl • Phenol: chloroform Prococol 1

(1: 1,

v/v) as m

• MtcrO-InJeCtton equtpment for Xenopus oocytes (see ref. 16)

Method 1 . Inject 25- 50 nl of the oligonucleotide solution (50 ng) mto the nuclet of at least 20 oocytes.

2.. Incubate the injected oocytes for 11 - 20 h at 20 ° C tn modified Barth' s buffer. 3. Repeat step 1. 4. Incubate the oocytes for 6-14 h as in step 2.

Contmued 168

Cathleen Enright and Barbara Sollner-Webb Proto c ol 16. Continued 5. Mix sufficient [ cx·32P] UTP with the 1-2 mg/ml oligonucleotide solution to give a final concentration of 5~-JCi 1-11 in the 32P-Iabelled UTP/oligonucleotide mixture. 6 . Inject 50 nl of the 32 P-Iabelled UTP/oligonucleotide mixture into the nucleus of each •OOcyte. 7. Incubate the oocytes for 6-14 h as in step 2. 8 . Homogenize the oocytes in extraction buffer. 9. Incubate the homogenate for 30 min at 50 °C.

10 . Extract the nucleic acids with phenol:chloroform and then wath chloroform alone; finally ethanol precipitate the extracted nucleic acids (see Protocol 1 s.teps 6-9).

11. Dissolve the nucleic acid in H 2 0 and store it at -70 °C. 12. Assay the RNA prepared from the macro-mjected oocytes for alterations 1n the abundanCE! of individual rRNA processang intermediates. This is conveniently done by electrophoresis (see Protocol 7, steps 5-9). Alternatively. primer extension, nuclease S 1, or Northern blot analyses (as described in Volume I, Chapter 2. Section 3) can be used to assess the extent of removal of the targeted snRNA. • Based on ref 16.

5.3 Immunological analysis of polypeptides required

for

processin~:

In addition to detecting snRNAs involved in processing, the presence and role of snRNP proteins can also be monitored and analysed. At present, there are no reported antibodies specific to individual nucleolar snRNPs. However, antibodies to fibrillarin, an -34 kDa polypeptide common to the U3, U8, U 13, and U 14 family of snRNPs, are available (reviewed in ref. I). These antibodies have been used both to immunodeplete the mouse S-100 extract of 5' ETS processing activity and to immunoprecipitate the processing complex that forms on pre-rRNA added to a processing reaction (34). For details of these procedures, see ref. 34 and Volume: I, Chapter 5, Section 6.

6. rRNA processing associated with transcriptional

termination Mention should also be made of two other cases of rRNA processing which have been found associated with, or part of, transcription termination in mouse and Xenopus laevis. TI1ese are summarized below (Sections 6.1 and 6.2), but protocols for their investigation are not reproduced here because these events seem better studied in 1the context of polymerase I transcription termination than of rRNA processiing itself. Furthermore, little is yet known about the 169

5: Ribosomal RJ\'A processing in \'ertebrotes proce,sing a'opect of these events. and so it seems advi~able that the invel>tigator should con~ult the entire original article prior to conducting studie~ on the\e topics.

6.1 3 ' end-processing in the mouse Coupled to the actual polymerase I transcriptional termination event is a very rapid proce,.,ing of the 3 '-end of the rRNA (17), \\hich ytelds what is generall~ recognized as the primary transcript. It appears that the t\\O event~ can be partl~ uncoupled when catalysed in vitro using fractionated nucleolar extracts or mutated rR'\A templates. The fact that the 3 -end of the processed product appear~ to map to the beginning of the first stem-loop structure preceding the termmation signal rather than to a particular nucleotide sequence (39) suggem the involvement of a 3 · exonuclease in the processing.

6.2 3' end-processing in Xenopus Jaevis In Xenopus laevis, the expected site of termination for rRNA gene transcription differs by one nucleotide from the functional termination site m X. boreulis (18). This mutated X. laews site, called T2, directs formation of the actual3 -end by degradation of downstream rR~A sequences (18). There appear~ to be a requirement for a functional downstream termination site in order that T2-directed 3 · end-formauon occur~. Further studie' are needed to determine the relationship, if any. of this processing to the termination-associated 3 ' endproces.,ing in the mouse described in Section 6.1

Acknowledgements The authors thank Drs Ted Gurney and Tim Nilsen for allowing them to pre~cnt their data and Ms Linda Miller and \1s \lary Bonkowski for preparation of the manu~cripr. Th1s work was supported b} ASC grant N-731B (to B. S ., .. ) and !\IH postdoctoral fellowstup (to C.E.).

References I. Sollner-\\ebb, B., Tyc, 1\.., and Stenz, J. (1993). In Ribosomal RNA: structure,

e1•o/ution, procesmrg und functron

2.

3.

4. 5. 6.

111 protein synthesis (ed. R Zimmerman and A. Dahlberg), CRC Pres~. Boca Raton, FL. (In press). \lu~ters, \\ , Venema, J., van der Linden, G., Van Heerikhuizen. H .. K1oot\\ijk. ) .• and P1anta, R. J (1989). Mol. Cell. Bioi., 9, 551. Mu.,ters. \\ .. Plama. R. J . 'an Heerikhuizen, H .. and Raue. A. (1990). In The nbosome-strucwre. junction and evolution. (ed. W. E. Hill et a/.), p. 3435. Am. Soc. Mtcrobtol., Washington DC. Li, H. V., Zagorski, J., and Fournier, M. J . (1990) Mol. Cell. Bioi., 10. 1145. Hughes. J 'vi. X. and Ares, M. Jr. (1991). E.\1BO J., 10, 4231 ToUcrve). D , Lehtonen, H., Carma-Fonseca, M., Hun, E. C. (1991 ). EMBO J.. 3, 5iJ .

170

Cathleen Enright and Barbaro Sollner-\\'ebb 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

24. 25 . 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40 41.

\\are,\. C., Renhawirz, R., and Gerbi, S. A. ( 1985). Nucl ACids Res, 13. 3581 Maden, B. E. H. (1990). Prog Vue/. ACid Res. and :Wol. Bioi., 39, 241. 1\liUer, K. and Sollner-Webb, B. (1981). Cell, 27, 165. Kass, S., Craig. N .. and Sollner-Webb, B. (1987). Mol Cell. Bioi., 7, 2891. Schumard, C. M. and Eichler, D. C. (1988) . J. Bioi. Chem., 263. 19 346. Hannon, G. J., 1\larone), P. A., Branch, A., Benenfield, B. J., Robemon, H. D .. and Nilsen, T . W. (1989). \.fol. Cell. Btol.. 9, 4422. Vance, V. B., Thompson, E. A .. and BO\\man. L. H. (1985). 'Vue/. Acids Res., 13, 7499. Schumard, C. M., Torres, C., and Eichler, D. C. (1990) . .\tfot Cell. Bioi., 10, 3868. Schumard, C.l\1., Torres, C., and Eichler, D C. (199 1). '.lot. Cell. BIOI., 11 ,593. Savino, R. and Gerbi. S. A. (1990). £.'.1BO J., 9. 2299. Kuhn. A. and Grumml, I (1989). Genes and De~·., 3, 224 Labhart, P. and Reeder, R. H . (1990). Genes and Dev., 4, 269. \\ellauer, P. K. and Dawid, l. B. (1974). J. Mol. Bioi., 89, 379. Wellauer, P. K. and Dawid, 1. B. (1974). J. Mol. Bioi., 89, 397. Forsheit, A. B.• Davidson, N., and Brown, D . D. {1974). J. Mol. BIOI., 90, 301. Gurney, T. Jr. (1985). Nucleic Acids Res., 13. 4905. Ausabel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G .. Smith, J . A., and Struhl, K. (1991). Current P•'Oiocols in molecular biology. \\ iley lnterscience, Ne'' York. NY. Gurney. T. Jr., Sorenson, D. K.. Gurne}', E. G., and \\'ills, M. (1982). Anal. B10chem., 125, 80. Maser, R. L. and Calvet, J.P. (1989). Proc. !'vall Aead. Set., USA. 86. 6523. Stroke, I. L. and Weiner, A. M. (1989). J. Mol. Bioi., 210, 497. Miller, 0. L. Jr. and Beatty, B. R. (1969). Science, 164, 955. Osheim, Y. N., Miller, 0. L. Jr., and Beyer, A. L. (1985). Cell, 43, 143 . Yip, M . T . and Holland, M. J . (1989). J. Bioi. Chem., 264, 4045. Peattie, D. A. {1979). Pror:. Nat/ Ar:ad. Sci, USA, 76, 1760. Craig, N., Kas~. S. and Sollner-Webb, B (1991). 'viol. Cell. B10l., II , 458. Dignam, J.D., Lebowitz, R ~>.( .. and Roeder. R. G. (1983). Vucleie Acids Res., II , 1475. Craig, N., Kass, S., and Sollner-\\'ebb, B. {1987). Proc. l\a/1 Aead. Set., USA., 84, 629. Kass, S. and Sollner-\\ebb, B. (1990) . •\lot Cell. Btol., 10, 4920. Bradford, 1\1. M. (1976). Anal. Biochem., 72, 248 Bartholomew, B .. Dahmus, M. E., and Meares, C. r. (1986). J. Bioi. Chem., 261, 14 226. Laemmli, U. K. (1970). Nature, 270, 680. Kass, S., Tyc. K.. Steitz, J. A .. and So liner-Webb, B {1990). Cell, 60, 897. Henderson, S., Ryan, K., and Sollner-Webb, B. (1989). Genes and De1· , 3, 212. Mougey. E B., Pape, L. K. and Sollner-\\ebb, B. (1993). Mol. Cell. Bioi.. 13 (In press). \1ougey, E. B.• O'Reilly, \1.. O~heim, Y., \tiller, 0. L.. Be}er. A. and Sollner\\ebb, B. (1993) Genes and De~·.. 7 (In pre~s)

171

Processing of transfer RNA precursors CHRIS L. GREER

1. Introduction 1.1 General methods T his chapter covers practical strategies and methods for studying the biochemistry of transfer RNA processing. Although processing of nuclearencoded tRNAs in yeast provides the primary focus for the chapter, essentially all of the strategies and many of the experimental methods apply equally well to the tRNAs of other organisms. This subject is generally amenable either to biochemical or genetic analyses. The methods in this chapter emphasize the biochemical approach and include the development of in vitro assay systems and characterization of reaction intermediates and products. Genetic methods for studying pre-tRNA substrate requirements are reviewed in ref. I and genetic approaches to the identification of trans-acting components are reviewed in ref. 2. Certain general methods applicable to tRNA processing, including fractionation of cellular RNAs, characterization of RNAs, a nd analysis of RNAprotein interactions, are described elsewhere in this book (Volume l , Chapters I, 2, and 5, respectively). The study of tRNA processing is, in many ways, unique within the RNA processing field. This assertion is based on both the unique physical and functional properties of tRNAs and the degree of complexity characteristic of tRNA biosynthetic pathways. Among the unique properties of transfer RNAs are their small size, stability, and relative intracellular abundance. These features make tRNAs attractive experimental subjects among cellular RNAs and the result has been the accumulation of a wealth of information regarding tRNA structure, function, and biosynthesis (see refs 3 and 4 for general reviews, and the compilation of tRNA and tDNA sequences in ref. 5).

1.2 tRNA structure and function The tRNAs function primarily in mRNA translation. Evolutionary constraints imposed by this crucial function are responsible in part for the high degree of conservation observed among tRNAs (reviewed in ref. 6). With the exception of certain mitochondrial examples, tRNAs share extensive common features

6: Processing of tra nsfer RNA precursors r.·

3'

E) - - Leader

/

e - -® Trailer

u Arm

D Arm

Ar m AC

Arm

Se quenc e Figure 1. Consensus pre·tRNA structure. The tRNA numbering scheme shown here is based on that descnbed in ref. 3. S1tes of variation in nucleotide sequence length occur in the 5' leader 3' trailer, and aotervening sequences. Invariant bases are represented by boxes lnd sem•·anvariant positions by ellipses. Positioos w1than the intervening sequeoce, S'leader end 3' tra1ler segments are Indicated by a colon, m•nus or plus sogn. respectJVely and are numbered from the adjacent exoo position. Solid hnes deoote segments of varyang length, N. The AA arm and AC arm mark the aminoacyl acceptor and anticodon arms, respectively. Arms commonly containing dihydrouridine and ribothymidine modif•cat1ons are marked D arm and T arm, respectively.

of primary, secondary, and tertiary structure. The primary sequence of mature tRNAs varies from 60-95 nt in length -with 13 imariaot and 12 semi-invariant position!>. E'\ten!>ive post-transcriptional modification is common with as many as 201t'o of the nucleotides of a mature tRNA species pre~ent in modified form (reviewed in refs 7, 8). A total of 52 different modifications have been characterized thus far, with some common to a majority of tRNAs and others unique to one or a few species (see the compilation in ref. 5). This facet of tRNA 174

Chris L. Greer structure greatly increase~ the number of di~tinct proces~ing reaction~ reqmred for the synthesis of any 'ingle tR "A species and also requires a rather large number of diverse processing pathways to produce the complete cellular repertoire of tRNAs. At the le,el of secondary structure. all of these tR'lAs form a consen·ed set of stem and loop structure which can be arranged 10 the traditional do,erleaf configuration. Thts structure and the corresponding nomenclature are summarized in Figure I. Significant length vanalion occurs only within the D-loop and variable arm segments \\ith the lengths of the remaining segments being highly consened. At the level of tertiary structure, aU tRNAs are thought to adopt an L-shaped configuration (reviewed in refs 9, 10). The small arm of the L-shaped ~tructure is formed by the aminoacyl acceptor (AA; Figure I) and T-stem helices, while the long arm consists of the D-stem and the anticodon stem and loop (AC; Fi~ure /). Tht~ conserved tertiary structure ts 'itabilized both by stad.ing interactiom between conserved helices and by tertiary interactions between conserved bases of the D- and T-loop segments, and the variable arm and D-stem regions. Thts high degree of consen·ation in both primary sequence· and threedimensional structure has two important implications for tRNA processtng. First, general processing enzymes may recognize their sub~tratte~ through conserved features within the mature, tRNA-like portion of the precursor, potentially at a distance from processing sites. Second, umque processing steps may requtre fine distinctions among tRNAs, potentially invoh ing discrete positive and negauve discnminators analogous to the determinants of tRNA identity in aminoacyl synthetase interactions ( II).

1.3 Overview of processing pathways The primary transcripts of tRNA genes m both prokaryote~ and eukaryotes must undergo a number of processing reactions to produce the mature, functional form of the molecule (12, 13). These reactions fall into three general categories: • removal of extraneous 'iequences • addition of non-encoded bases • introduction of modifications to the base and ribose moieties of specific nucleotides The temporal order of the~e processing events has been examined in a number of prokaryotic and eukaryotic systems. In all cases, there appears to be a preferred sequence of events for a given precursor since onl) a subset of the possible mtermediates has been detected. For example, processing in Xenopus oocytes is thought to start with trimming of the 5 '-end followed by maturauon of the 3 -end , splicing, and finally transport out of the nucleus (14). The addition of individual base modifications occurs at distinct stages in tills size-maturation process ( 15). While a similar order rna) apply to some precursors in yeast, others 175

6: Processing of transfer RNA precursors

may utilize different pathv\ays (16). Whether substrate specificity, functional compartmentation, kinetic constraints, or a combination of these facwrs define the processing pathways for individual tRNAs remain among the most pressing questions in tRNA bios)'"Tlthesis.

1.4 Biochemical analysis of tRNA processing The rest of this chapter is divided into three experimental sections intended to reflect the approximate temporal order one might follow in initiating a biochemical study of a tRNA processing reaction. Section 2 describes the preparation of tRNA precursors for use as substrates in processing reactions. Precursors may be purified from whole cells or be prepared by m vuro transcription. Section 3 includes the preparation of cell-free extracts active in transcription and specific processing reactions. Examples of the preparation 1 ~ complex nuclear extracts and purified splicing enzyme fractions are provided Section 4 describes the development of tRNA processing assays and provide\ examples of assays for measuring changes in the size and sequence of tht: precursor or for detecting the introduction of specific nucleotide modifications

2. Preparation of pre-tRNA processing substrates 2.1 Introduction A biochemical approach to the study of tRNA processing relics heavily on the provision of substrates of high purity and defined structure, and usually in large amounts or at high specific radioactivity. Additional requirements are the facility for manipulating substrate sequence or structure, the potential for site-speciftc labelling, and the ability to include or omit one or more of the specific processmg steps in preparing the substrate. Two general approaches are available fo r the preparation of processing substrates: • expression in vivo followed by isolation from whole cell RNA fraction • expression in vitro using homologous or heterologous transcription/ processu.g systems Each of these approaches has distinct advantages and limitations which should be considered when choosing an appropriate experimental strategy. These are described along with the methods themselves in the sections below

2.2 Preparation of nuclease-free carriers Labelled RNA substrates must often be concentrated from dilute solutions by precipitation with ethanol. At RNA concentrations less than 5 ~g mi. precipitation may be inefficient unless an appropriate carrier is added. For samples to be subjected to nuclease digestion (see Section 4 .3), the addition of a known amount of RNA carrier is appropriate. For samples to be used in processing or post-labelling reactions, glycogen should be added as carrier. Protocol/ describes the preparation of nuclease-free carriers for these purpose-. 1 76

Chris L. Greer Prot ocol 1 . Preparation of nuclease-free RNA and glycogen carriers

Reagents • 2. 0 M sodium acetate buffer pH 5.0 e TE buffer !1 0 mM Tns- HCI. pH 7 5. 1 mM EDTAl e 20'lb SDS

e s.O M NaOH e Proteonase K 120 U .mg protem, Sogma, p 65561

• Phenol chloroform : 1soamyl alcohol 125 24 : 11 • Sterile water; treat deoomzed dosulled water with 2 drops per htre of DEPC, autoclave 1 h, 120 °C, 16 p.s.i. • Either glycogen !oyster glycogen Sogma Type II G8751 l or unfractionated RNA from yeast ISigma Type VI R6625 o

Method 1. Dissolve the glycogen or RNA 1n TE buffer to 20-1 00 mg/ml final concentration. Note that this form of RNA must be carefully neutralized to d1ssolve it; add 5 M NaOH dropw1se with st1rnng. Use indicator strips to check that the solution is at pH 7. 5. 2. Add SDS to 0. 5%, proteinase K to 1 mg ml and incubate the solution for 30 min at 55 °C. 3. Extract the solution with 1 vol. of phenol. chloroform: isoamyl alcohol. Centrifuge at 2000 g for 5 min to separate phases. Repeat the extraction of the aqueous phase twice more and finally extract it with 1 vol. of ether. 4. Add 0. 1 vol. of 2 M sodium acetate buffer and 2 vol. of ethanol. Keep the mixture on crushed dry oce for 1 h to precipitate the RNA or glycogen.

5. Centrifuge for 15 min at 12 000 g, 4 °C. 6 . Air dry the pellet, resuspend it in 0.1 vol. of 2M sodium acetate buffer, and repeat the ethanol precipitation (steps 4 and 5) twice. 7 . Resuspend the dried pellet in sterile water to 20 mg/ml final concentration and store the sample at - 20 °C in 1 ml aliquots.

2 .3 Preparation of tRNA substrates from whole cells This approach is especially applicable where only limited information exists regarding the particular substrates or processing reactions to be studied and where an initial characterization i~ required. Substrates prepared in this way may have already undergone some processing steps, such as the introduction of base modifications, which might be required for the processing reaction to be studied. If so, analysis of the partially-processed species may provide insights into the nature of the naturally-occurring substrates. Disadvantages to this approach include the difficulties in obtaining highly purified fractions, the limited strategies available for specific radiolabelling of substrates either during in vivo synthesis or after their isolation, and the low levels of tRNA precursors normally present in ceUs. This Iauer difficulty may be partially overcome through the use of mutant cell lines or altered growth conditions \\Chich result in the 177

6: Processing of transfer RNA precursors accumulation of panially-processed transcripts. An example of this approach relies on the phenotype of the rnal-1 mutation in yeast (17): the accumulation of imron-comaining tR'\A precursors upon shift to the re:.tricti,·e temperature. This method (ba,ed on ref. 18) is de cribed here in t\\O part~: growth ana labelling ol the yeast cells (Protoco/2) and the extraction and i'olation of tRNA precursors (Protocol 3 ). The procedure relies on phenol e\.traction of nuclei<. acids and their purification by gel electrophoresis; general re\lews of these methods can be round in refs 19-21. Alternative procedures for tRNA purification include conventional chromatography and HPLC as summarized m refs 22 and 23. Prot ocol 2. Growth and labelling of cells Reagents • Saccharomyces cerevtstae. stra1n M304; rna 1/mal, ade1lADE1, ade2 ADE2 his1 HIS7 ,/ys2 LYS2, cyr1 TYRl CAmi!rican Type Culture Collect1on 1142245) •

32 [ P) -orthophosphate CHCI-free, camerfree, 40 mC1-ml; Amersham cat. no. PBS 431

• Bactopeptone yeast extract. and yeast nitrogen base With ammo acids (Dileo; catalogue numbers 0118-01-8, 0127-01 7, and 0392-15, respectively) • 20% glucose; sterilize 45 min. 120 °C, 16 p.s 1.

e SUCGiniC aCid (solid) e 1.01M MgCI • Sterile water !Protocol II • YPD Inch medium)•. For 1 litre culture, add 10 g yeast extract, 20 g Bacto· peptone to 900 ml of deionized water; sterilize by autoctav1ng 1 h. 120 °C, 16 p.s.i.}; add 100 ml of 20% glucose just before 1noculatmg with yeast

• YPD agar plates las for hqu1d med1um above With the add1t10n of 20 g agar per litre of medium) • 10" succmate stock solution. 01ssolve 100 g succ1nic acid, 50 g NaOH in 1 htre of deionized water, adJuSt the pH to 5.7 and autoclave 11 h, 120 °C, 16 p.s.LI • 10" YP stock solut10n. D1ssolve 100 g Bactopeptone, 50 g yeast extract, 67 g yeast nitrogen base with am1no ac1ds in 750 ml of deion1zed water Add cone NH4 0H (14.8 Ml slowly to pH 10. Add 100 ml of 1 M MgCI 2 and mcubate at 4 °C, 8-10 h to preCIPitate free phosphate. Add 50 ml of 1 M MgCI 2 and incubate 4 h at 4 °C Remove the precipi tate by centnfugat1on at 3000 g for 15 m1n Carefully decant the supernatant and add cone. HCI to pH 6.5. Adjust the fmal volume to 1 litre with water and autoclave 11 h, 120 °C, 16 p.s.i.l • Low-phosphate labell1ng medium. Mil< 25 ml of 10 x succ1nate stock solution, 25 ml of 10 x YP stock solution. 25 ml of 20% glucose, and 175 ml of sterile water

Method

NB Tfle labelling involves the use of a large amount of 3 2p radioactivity. Take care in its handling and disposal. 1 . Start a culture of yeast strain M304 by moculat1ng 5 ml of YPD medium w •th a colony from a fresh YPD plate. Grow overn1ght wtth shaking at

24 °C

Continued

178

Chris L. Greer Protocol 2 . Continued 2. Add the 5 ml overnight culture to 200 ml of fresh YPD medium and mcubate at 24 °C to A 660 :0: 1.0. 3. Collect the cells by centnfugmg at 7000g for 5 mrn at 4 °C Wash the cell pellet by resuspending the cells in 50 ml of low-phosphate labelling medium and centrifuging as before 4 . Resuspend the washed cells m 200 ml of fresh low-phosphate labelling medium and incubate with shaking for 3 h at 24 °C . 5. Add 50 mCr of [ 32P]orthophosphate. Incubate for 5 mrn at 24 °C. 6 . Carry out a temperature shift by transferring the culture to a shakrng water bath at 37 °C. 0 Incubate with shaking for 30 mrn. 7 . Chill the culture for 5 min in an ice-water bath. 8. Collect the cells by centrifugmg at 7000 g for 5 min at 4 °C . Carefully remove all the supernatant from the cell pellet. • Additoonal information regarding yeast growth media can be found rn ref. 24 • Use of a water bath os essenttal to achoeve the rapod increase on temperature necessary for induction

Protocol 3 . Extraction and purification of labelled tRNA precursors from yeast cells

Equipment and reagents eDEAE-cellulose column; Whatman DE-52 (4057 -200). 0.5 ml equilibrated rn column loading buffer and packed rnto a disposable column or synnge • Ouik-Sep columns (disposable, lsolab. code OS ·Yl • Sterile water !Protocol 11 • NES buffer (0. 1 M sodoum acetate, pH 5.7 20mM EDTA 1% SDSI • 2.0 M ammonrum acetate. pH 7 .0

• First-dimensronal gel ( 10% ac:rylamode, 0.33% bosacrylamide, 4 M urea on TBE buffer, 31 x 0.2 em) and second-domensronal gel (20% acrylamode. 0 66% bosacrylamrde, 4 M urea on TBE buffer· 31 x 41 x 0.2 cml plus extra seconddimensional gel mixture, and electro phoresis equipment (see Volume I, Chapter 1, Protocol 81 • Ammonium persulphate 10%. aqueous solutron, made fresh daoly l

• Redostolled phenol, saturated wrth 20 mM Tns-HCI, pH 7 , 4

e TEMED (IBIInc .. 701201

• Column loading buffer (0 2 M NaCI, 10 mM T11s-HCI, pH 7.41

• Glycogen carrrer, prepared as on Protocol 1

• Column elutoon buffer ( 1 5 M NaCI 10 mM Tris - HCI, pH 7.41

• Gel elution buffer (0 .3 M NaCI. 20 mM Hepes-KOH. pH 7.4, 1 mM EDTA, 1% phenoH

• TBE buffer (90 mM Tns-borate. pH 8.3 , 2.5 mM EDTAI • Gel sample buffer (20% sucrose, 7 M urea, 0 . 1% bromphenol blue, 0. 1% xylene cyanoH

e 10mM Tris- HCI, pH 7.4 • Yeast cells labelled with phosphate from Protocol 2

I 31P ]ortho·

Contmued 179

6: Processing of transfer RNA precursors Protocol 3.

Continued

Method 1 . Resuspend the yeast cell pellet in 4 ml of NES buffer. Add 4 ml of phenol and mix by vortexing. Incubate at 55 °C for 30 min, vortexing at 10 mm intervals 2 . Separate the aqueous and phenol layers by centrifugation (2000 g. 5 min at room temperature). 3

Set aside the aqueous fractton. Add 4 ml of fresh NES buffer to the phenol layer and repeat the extraction as 1n steps 1 and 2

4. Combme both the aqueous layers and extract them w1th 8 ml of fresh phenol for 20 mm at room temperature (steps 1 and 2} . 5 . Transfer the re-extracted aqueous layer to an appropriately sized centrifuge tube, add 12 ml ethanol and keep for 4-8 h at - 20 oc. 6 . Collect the RNA precipitate by centnfugmg at 17 000 g for 30 min at 4 °C. Resuspend the pellet in 4 ml of column loading buffer 7 . Apply the redissolved RNA to the equilibrated DEAE-cellulose column Wash the column w1th 10 ml of column load1ng buffer 8 . Elute the column with 4 ml of column elution buffer. collecting the ent1re eluate m an appropriately sized centrifuge tube. 9 . Add 3 ml of water, 1 ml of 2.0 M ammonium acetate buffer and 20 ml of ethanol. Keep the mixture on crushed dry ice for 1 h.

10. Centnfuge at 17 000 g for 30 min at 4 °C . D1scard the supernatant and wash the RNA pellet by adding 5 ml of 70% ethanol and centrifuging at 12 OOOg, 15 min. 11 . Air-dry the pellet and resuspend it in 10 ~I water.

12. Add 20 ~I of gel sample buffer. 13. load the sample on to the first-dimensional gel. 14. Using TBE as the electrode buffer, run the gel at 40 rnA (constant current} until the xylene cyanol marker dye has migrated 25- 30 em ( 16-20 h)

15. Using a stenle razor blade, cut out a 20 em stnp of the sample lane from 5 em ahead of the xylene dye to 1 5 em behind 1t.

16. lay thts strip horizontally across the top of the second-dimensional gel 17. Add 2 ml of ammontum persulphate and 50 ~I of TEMED to 250 ml of second-dimensional gel mixture. Use th1s to cast the strtp into place on top of the second -dimensional gel.

18. Electrophorese the second-dimensional gel at 2 50 V until the xylene cyanol marker reaches the bottom of the gel (- 20 h). At th1s point, RNAs 30- 70 nt m length should be well resolved in the middle of the gel. For better resoluuon of products 70-120 nt in length, add a fresh aliquot (25 ~I} of gel sample buffer to the top of the gel to provide a trackmg dye and continue the electrophoresis until the xylene cyanol marker again reaches the bottom of the gel.

Continued 180

Chris L. Greer Protocol 3. Continued 19. D1sassemble the electrophoresis apparatus leav1ng the gel on the glass backing plate. Wrap the gel and plate in plastic cling film and mark the s1des and corners with radioactive or fluorescent mk for later alignment of gel and f1lm. 20. Visualize the RN1~ bands by autoradiography (e.g 8-1 2 h exposure at -70 °C using Hyperfilm-MP and an intensifying screen). Develop the film according to the manufacturer's instructions. 21 . Consult refs 25 .and 26 for a comparison of patterns, but m general pre-tRNAs and their processed products m1grate off the diagonal formed by random RNA fragments. Cut out mdiv1dual bands using a clean razor blade and elute the RNA by crushing each gel slice in a sterile glass v1al using a glass rod and soakmg the gel fragment in 2 ml of gel elutiOn buffer at 37 °C for 10--12 h. 22. Remove the gel fragments by filtenng through a Ouik-Sep column. 23. Recover the RNA by addmg 0.4 ml of 2M ammonium acetate buffer, 20 ~o~g glycogen earner, and 7 5 ml ethanol and then proceeding as in steps 9-11. 24. Resuspend the fmal pellet m 20-50 ~o~l of 10 mM Tris-HCI, pH 7 .4, and store it at -20 "C.

Labelled pre-tRNA substrates prepared as in Protocol] have a high specific radioactivity (5-6 x 10'5 d.p.m.IIJg) for use in assays requiring very high sensitivity. For applications which tolerate reduced sensitivity, the amount of label used can be reduced accordingly. Unlabelled pre-tRNA can also be prepared by this method, visualizing the products in the gel (step 20) by staining with ethidium bromide (0.5 pg/ ml in TBE buffer). In this case, remove the ethidium bromide from the eluted! RNA (step 23) by extracting twice with an equal volume of propan-2-ol. Pre-tRNA substrates prepared by this method contain many, but not all, of the base modifications characteristic of the mature tRNA product. For example, pseudouridine (.jt), ribothymidine (T), and dihydrouridine (D) are present at high yield in pre-tRNA Tyr, while only socro of the expected 1-methyladenosine (m 1A) is detected (ref. 27). Thus the characterization of each new substrate prepared by this method should include an analysis of base modifications (see Section 4 below).

2.4 Preparation of substrates by in vitro transcription This approach is applicable where a cloned t RNA gene is available and where requirements for other processing steps are either defined or can be taken into account in the expression system chosen. There are a number of distinct advantages to this approach including: • the ability to label the RNA substrate in defined nucleotide positions and to selected specific radi,oactivities 181

6: Processing of transfer RNA precursors

e the ease of preparing pure substrates with defined structure • the ability to produce rare intermediates, aberrant products, and substrate analogues by manipulating the structure of the transcription template e the ability to incorporate nucleotide analogues, such as photoreacti\ e or biotinylated derivatives, for use in cross-linking or rapid affinit y-based purification procedures Both homologous and heterologous in vitro tRNA gene expression systems are available. The homologous systems rely on expression by RNA polymerase III and intermediates can be produced by processing activities present in extracts of whole cells or nuclei. This latter feature, including the presence of certain base-modifying activities in some extracts, represents one advantage to this approach. A significant limitation is that tRNA gene sequences required for transcription and other processing steps cannot readily be altered. Examples of such coupled transcription/ processing systems are described in Section 3 below. Heterologous systems are primarily based on the use of a plasmid DNA template containing a bacteriophage promoter fused to a tRNA gene and purified phage or bacterial polymerase for in vilro transcription reactions. Examples from the literature of the use of SP6, lambda, and T7 bacteriophage promoter fusions to produce tRNA processing substrates are provided in Table 1 and these should be consulted in choosing a specific expression system. These heterologous systems have a number of distinct advantages: (a) The presence and composition of 5' -leader and 3 ' -trailer tRNA sequences can be controlled. In the former case, this is achieved by appropriate positioning of the phage promoter in the original gene construction and in the latter by the choice of the restriction site used for transcription 'run-off' . (b) Transcribed sequences can be altered at will without concerns for expression requirements characteristic of homologous systems. (c) Transcripts can be prepared in high yield with these efficient transcription systems. (d) Incorporation of labelled or derivatized nucleotides can be controlled through the selection of appropriate nucleotide substrate mixtures. Limitations to this approach are the inability to modify bases and rhe formation of substrates with inappropriate conformations under the in vi1ro transcription reaction conditions. The former problem can be addressed in some instances by incubation of the substrates in processing extracts (see Volume 1. Chapter I). The latter problem may be alleviated by reannealing the purified substrates prior to their use in processing reactions (28). An example of the use of T7 polymerase to produce pre-tRNA Phe substrates labelled using [ ~- 32 P] UTP is described in Protocol 4. Efficient transcription requires ~ 50 IJM (final concentration) of each of the elongating nucleotides and > I 00 IJM of the 182

Chris L. Greer initiating nucleotide (usually GTP). The low UTP concentration u-;ed in Protocol 4 reduces the reaction yield but results in high specific radioactivities

(- 5 x 103d.p.m./fmol).

Table 1 . Examples of phage promoter/tRNA gene fus1ons for the production of semisynthetic processmg substrates Applications

Promoter type/tRNA gene

References

Phage SP6 promoter

B

54

Schizosaccharomyces pombe tRNA 5 ••

A

55

Saccharomyces cerevisiae tRNALeu

A

56

Escherichia coli tRNAPhe

A

57

C,D, E

28,58

Saccharomyces cerev1s1ae tRNA Giu (mitochondrial)

Phage A promoter

Saccharomyces cerevisiae tRNA Tvr Phage T7 promoter

Saccharomyces cerevisiae tRNAPhe

D, E

59,60

Mycoplasma mycoides tRNAGiv

c

61

Schizosaccharomyces pombe tRNA5 ••

A

62

• uses made of the semisynthetic substrates 1n the references listed include: lA) formation of mature 5'-ends; (B) processing of 3'-ends; IC) addition of one or more specific nucleotide mod1f1cations; (D) presence of intron endonuclease and/or tRNA ligase activity; and (E) structural analysis.

Protocol 4 . In vitro tRNA gene transcription using T7 RNA polymerase Equipment and reagents • Phage T7 RNA polymerase 160 U/~1. Pharmacia 2 7-080 1-01 ) • Ribonucleotide stock solutions; separate 0. 1 M stock solutions in 10 mM TrisHC), pH 7.5 (ATP, 27-2056-01; CTP, 27-2066-01; GTP, 27-2076-0 1, UTP, 27-2086-01; Pharmacia) • [a·32P[UTP (10 mCi/ml, 3000 Ci/mmol; Amersham, PB. 10200-PB. 10203)

• Plasmid DNA containing the tRNA gene cloned next to a T7 phage promoter; prepared using QIAGEN cartridges (Oiagen 51 003) and the procedure recommended by the manufacturer • 5 x transcnpt1on buffer (0.2 M Tns-HCI, 50 mM MgCI 2 , 50 mM NaCI) • TE0.1 buffer (10 mM Tris-HCI, pH 7.5, 0.1 mM EDTA)

Continued

183

6: Processing of transfer RNA precursors

Protocol 4 . Continued • 50 mM OTT Store at - 20 °C for up to 1 week • Stop moxture 10.1 M EDTA, pH 8 0. 2% SDS. 2 mg lml proteinase K !Sigma, P

65561 • Phenol : chloroform: isoamyl alcohol

• Glycogen earner (20 mgtml) and sterile water prepared as 10 Protocol 1 • TBE buffer, 2.0 M ammonoum acetate, pH 7.0, gel sample buffer. and gel elutoon buffer as In Protocol 3 • Appropriate restriction enzyme and buffer

(25·24:11 • Polyacrylamode gel (15.5% acrylamode, 0 5% bosacrylamode 8 M urea, 1 >< TBE buffer 19 >e 22>e0.075 em); and electrophoresis equopment (see Protocol 31

• 5 x rNTP moxture 12.5 mM each of A TP GTP, and CTP plus 0 .05 mM UTPJ • • 0.4 M ammonoum acetate buffer. pH 7.0

Method A. Restriction of plasmid DNA template 1. Linearize the plasmid DNA by restriction digestion at the appropnate transcription ' run -off' site.

2. Extract the DNA by vortexing w1th an equal volume of phenol: chloroform . isoamyl alcohol. Centrifuge (2 min) in a microcentrifuge at room temperature to separate the phases.

3 . Precipitate the DNA from the upper (aqueous) phase by adding an equal volume of propan-2-ol and leaving the mixture at - 20 °C for 0.5 h. 4. Recover the DNA by centrifugation in a m1crocentrifuge for 15 mm. 5. Air dry the pellet, redissolve It on sterile water at 1 (JQI (JI and store it at -20 °C. B. In vitro transcription of DNA template

1 . Prepare a 50 (JI transcription reaction in a microcentrifuge tube on ice b r adding: • sterile H 2 0

12 IJI

• 5 x transcnption buffer

10 IJI

• on

10(JI

• 5 x rNTP mixture

10 (JI

• plasmid DNA template

11JI

e [a-32P] UTP

51JI

2 . Start the reaction by adding 2111 of T7 polymerase and 1ncubate for 1 h at 37 °C. 3 . Stop the reaction by the addition of 5 IJI of stop mixture and incubate for 15 min at 50 °C. 4. Add 40 IJI of 2.0 M ammonium acetate, pH 7 0, 1051JI sterile water 0.5 ml ethanol and keep the mixture on crushed dry 1ce for 1 h to precipitate the nucleic acids.

Continued 184

Chris L. Greer

Protocol 4 . Continued 5 . Centrifuge tn a microcentnfuge at 4 °C for 1 5 mm Wash the pellet once w ith 70% ethanol at 4 ° C . A ir dry the pellet and dissolve it in 5 ~I of sterile water. 6. Add 5 ~I of gel sample buffer and incubate for 5 mm at 65 ° C. 7 . Separate the RNA product by electrophoresis in the 15. 5 % polyacrylamide gel at 20 rnA until the bromophenol blue dye has migrated 17 em (2.0-2.5 h). Note that unincorporated nucleotides comigrate with bromophenol blue and that pre-tRNA transcnpts migrate behind the xylene cyanol marker Dtspose of the used electrode buffer as radioactive waste. 8 . Disassemble the electrophoresis apparatus leavtng the gel on the glass backmg plate. Wrap the gel and plate in cling film and mark the sides and corners with radioactive or fluorescent ink for later alignment of gel and film. 9 . V1suahze the RNA products by autoradiography (e.g. 5-15 min exposure using Hyperf1lm-MP and an intensifying screen) Develop the film according to the manufacturer's instructions. 10. Align the gel and film on a light box. W tth a razor blade, cut out the appropriate regton of the gel and elute the RNA from each gel slice by soaking it for 8 - 12 h at 37 °C in 2 ml of gel elution buffer as described in Protocol 3 (step 21 ). 1 1 . Add 0 .5ml of 2.0M ammonium acetate buffer, 2 . 5~1 of 20mg ml glycogen earner, 7 5 ml of ethanol, and prectpitate the RNA on crushed dry ice for 1 h. 12. Centnfuge for 20 mtn, 1 5 000 g at 4 °C. Dry the pellet and resuspend it in 0.35 ml of 0.4 M ammonium acetate buffer. 13 . Repeat the ethanol precipitation as in steps 9 and 10. 14. Wash the pellet in 70% ethanol. Resuspend the final dried pellet in TE0.1 buffer ( 10 000 c.p.m. ' ~I) and store at -20 ° C. • when labelling w1th a nucleoside tnphosphate other than [a- 32P] UTP, mod1fy the compos1t1on of th1s 5 x rNTP mtxture accordingly.

3. Preparation of tRNA processing extracts In addition to a method for preparing appropriate substrates, a biochemical approach to tRNA processing requires the ability to prepare active processing ext racts. Initially. the characterization of a processing reaction is often carried out with crude cell extracts. Long-term goals should include reconstitution of the reaction from highly purified components, obtained by fractionation of extracts from the original cell source or through the use of heterologous expression systems. The protocols described in this section represent examples from each of these three categories: preparation of a crude nuclear extract containing a variety of activities, partial purification of a processing enzyme from yeast, and the use of a bacterial expression system. 185

6: Processing of transfer R.\'A precursors Table 2. Sources of active tRNA processing extracts Activities

References

Escherichia coli

A,B,C

63-65

Bacillus subtilis

A

66

A. B

67

D

27

A,D

68

A

69

Saccharomyces cerevisiae

A B

54

Rat (liver)

A ,B

70

A

71

Extract source Eubacteria

Thermus thermophllus survey of diverse bacterial sources Archae bacteria Halobacterium halobtum Sulfolobus solfatancus Mi1ochondria

Human (Hela cells) Chloroplasts Euglena gracilts Spinach Ma1ze

A,B,C

72

A

72, 73

A ,B

74

A , B,C

31

Eukaryotes Saccharomyces cerevtsiae Schtzosaccharomyces pombe

A

55

Chlamydomonas

D

75

Wheat

A,B,C. D

A B

Drosophila melanogaster Bombyx mori

76

7~

79

A, B

80

Xenopus laevis

A,B,C,D

81-83

Human (Hela, KB. leukaemia cells)

A, B,C,D

84-88

Processmg steps exam1ned in the publications listed include: IAJ format1on of mature 5'-ends; fBI process1ng of 3'-ends; ICJ add1110n of one or more spec1fic nucleotide modifications. and (OJ presence of mtron endonuclease and ·or tRNA ligase activ1ty

186

Chris L. Greer

3.. 1 Preparation of crude cell extracts Crude or partially-purified extracts containing tRNA processing activities have been prepared from a wide variety of sources and the literature provides an excellent starting point in designing an experimental strategy. Table 2 provides an over,iew of this literature with examples from eubacterial, archaebacterial, eukaryotic nuclear, and eukaryoric organellar sources. Many of these extracts are active for transcription as well as certain processing reactions allowing their use for studying coupled synthesis and maturation reactions. Advantages and disadvantages in the use of these extracts are both rooted in their complexity. Thus, one advantage is the ability to examine processing steps requiring several components or multiple processing reactions. A clear disadvantage is the presence of competing activities, including nucleases, proteases, phosphatases, etc. H owever, with appropriate regard for the inherent limitations, these complex extracts can be used effectively to obtain useful information regarding specific tRNA processing reactions. Protoco/5 describes the preparation of a crude yeast nuclear-extract fraction and is adapted from the procedure described in ref. 29.

Protocol 5. Preparation of a nuclear extract from yeast Equipment and reagents • Oounce homogenizer (40 ml size) woth large (A) and small clearance (8) pestles IKontes, 885300-00401 • Oak Ridge type plastic centrifuge tubes (30 ml, capped I

• Saccharomyces cerevisiae straon JHRY20-2CO 1 (protease-deftcient•, MATa, his30200, ura3-52 . /eu2-3. 112, pep4-0 1 .. URA3; other proteasedefocient straons can be substotuted (e.g. 208- 12. Amerocan Type Culture Collection, 206261

• 1% SOS • Sl and Sll spheroplastlng buffers 1100 ml each). Prepare 200 ml of 1.0 M sorbitol, 10 mM MgCI2 , 50 mM Tris-HCI, pH 7 .8. Oivode thos into 2 ahquots of 100 mi. Just pnor to use add 3 ml of 1 0 M OTT to one aliquot to make Sl buffer and 0 .3 ml of 1.0 M OTT to the other aliquot to make Sll buffer • Sill lysos buffer (1 00 ml; 15 mM KCI , 5 mM MgCI 2 , 0 .1 mM EOTA. 10 mM Hepes - KOH pH 7 81

• 1.0 M OTI ( 10 ml); make up fresh using sterile water (see Protocol II

• Oialysos tubong and doalysos buffer (3 litres; 0 . 1 M KCI, 10% glycerol, 0 . 1 mM EOTA. 20 mM Hepes-KOH, pH 7.81. Autoclave (1 h, 120 ° C, 16p.s.i.l; add 3 ml of 1 0 M OTT JUSt before use

e YPD medium (Protocol 2 )

• Solid INH.12SO. and 4 .0 M INH 12SO.

• Yeast lytoc enzyme (1 0~ U 'g, ICN 8iomedocals 190- 123)

Method NB Carry out steps 7-3 at room temperature and all other steps at 2-4 °C.

1. Grow the yeast cells in 1 litre of YPD medium at 30 ° C with aeration until A 600 =6.0- 10.0. Harvest the cells by centrifugmg at 3500g for 5min.

Contmued

187

6: Processing of transfer RNA precursors

Protocol 5. Continued 2. Resuspend the cells m 33 ml of Sl buffer and d1v1de the suspens1on into two Oak Ridge-type cemtrifuge tubes. Centrifuge at 3500 g for 5 min 3. Resuspend each pellet in 16.5 ml of Sll buffer and add 1.6 mg of yeast lytiC enzyme to each tube. Incubate the cells at 30 °C with gentle agitation until spheroplasting is complete. Momtor the spheroplasting as follows. Before addition of the lytic enzyme and at 10 min intervals thereafter, remove a 10 JJI sample of cell suspension and add it to 2 ml of 1% SDS. After 2 min, measure the A 600 . Spheroplasting is complete when the absorbance falls to 20% of the time zero value (usually after 30-70 mini. Excessive or incomplete spheroplasting greatly affects the final activity of the extract. 4. Collect the spheroplasts by centrifugmg at 2000 g for 5 m1n at 4 °C Resuspend them gent I•( with a glass rod m Sll buffer ( 16.5 ml per tube I and repeat the centrifugation 5. Resuspend each pellet m 10. 5 ml of Sill lysis buffer. Combine the contents of the two tubes into the Dounce homogenizer (prechilled) and homogenize once gently with the A pestle. Incubate the suspension for 20 min on ice. 6. Lyse the spheroplasts bv homogenizing VIgorously 3 t1mes w1th the B pestle. 7 . Collect the particulate fract1on (includmg the nuclei) by centrifuging at 17 000 g. 10 min at 4 °C and carefully pour off the supernatant. 8. Resuspend the pellet in 8 ml of Sill buffer, transfer the suspension t o the homogenizer, and use an additional 2 ml of Sill buffer to rinse out the centrifuge tube. Homogenize the comb1ned suspension once gently w1th the A pestle. 9. Calculate the volume of the 'breakage pellet' (for use in step 11 below) by measuring the final volume of homogenate m step 8 and subtracting 10 ml (the total volume of buffer added). Add 0 .2 vol. of 4 .0 M (NH 4 ) 2 S0 4 . Extract fo1• 10 min on ice with occasional mixing.

10. Remove particulate material by centrifuging at 1 50 000 g for 1 hat 4 °C Remove the supernatant carefully and measure the volume. Add 0 .25 g/ml of solid (NH4 bS04 . Mix gently and mcubate for 10 m1n on ice

11 . Collect the precipitate by centrifuging at 17 000 g for 30 min at 4 °C Resuspend the pellet .n 0 . 5 times the volume of the ' breakage pellet (calculated in step 9).

12. Dialyse the suspension against one litre batches of dialysis buffer (making 3 changes of buffer at 1 h intervals) at 4 °C with stirring.

13. Transfer 50 Ill aliquots of the d1alysed extract to m1crocentrifuge tubes and store them frozen at - 70 °C. • See ref 30.

Typical yields from this procedure are 2-3 ml of an extract containing 8-12 rng total protein per millilitre. This extract is effic1ent in tRNA gene transcripuon (29) and is active in a number of processing reactions including 5 ' -end maturation, 3 ' -end trimming and CCA addition, and base modifications su ... ll as the formation of pseudouridine, dihydroundine, ribothymidine, and certa n 188

Chris L. Greer methylated derivatives (when supplemented with 0.1-l mM S-adenosylmethionine). The extract also contains a small amount of tRNA endonuclease and RNA ligase, but splicing IS inhibited at the salt concentrations typically used in transcription reactions (30). Thus end-mature, unspliced precursors accumulate in reactions using intron-containing tRNA gene templates. Accumulation of 5' and 3 end-extended precursors can be accomplished by carrying out transcription reactions at a reduced temperature (12-18 °C).

3.2 Partial purification of processing activities Deterntination of the reaction kinetics of RNA processing, the structures of proce sing intermediates and products, and the concentration dependence of substrate, enzyme, or co-factors is often not feasible in crude extracts due to the presence of competing activities and the poorly defined enzyme, co-factor, and RNA content of the extract. Although reconstitution from pure components may be the ideal way to investigate these aspects, the use of partially purified fractions provides a realistic intermediate step in the characterization of a processing system. Purification procedures are necessarily idiosyncratic and are developed empirically. However, a common strategy is the initial application of conventional protein fractionation techniques which rely on the general physical properties of the proteins of interest, followed by specific affinity methods which take advantage of distinct functional properties. Conventional techniques often applied to tRNA processing activities include differential ammonium sulphate precipitation, ion-exchange chromatography (particularly using heparin-agarose, phosphocellulose, DEAE-cellulose, hydroxyapatite, and certain FPLC resins, including Mono Q and Mono S from Pharmacia), and molecular sieving columns. Affinity chromatography methods include the use of immobilized tRNA or synthetic RNA resins, and tRNA-mediated elution from conventional resins (32, 33). Protocol 6 describes a partial purification of yeast tRNA endonuclease. A more extensive purification procedure can be found in ref. 34. The method described here leads on from the preparation of the nuclear extract described in Protoco/5 which should be consulted before proceeding. This method takes advantage of the observation that yeast endonuclease behaves as an integral membrane protein and is adapted, in part, from the method developed by Peebles eta/. (35). Note that in some experimental systems, such as Xenopus oocytes (36), the endonuclease may be found in the soluble fraction and so this purification procedure should be modified accordingly.

Protocol 6. Preparation of partially-purified endonuclease from yeast

Reagents • Yeast nuclear pellet prepared as described 1n Prococol 5, steps 1-7

• 1.0 M OTT (see Prococol 5l

189

Contmued

6: Processing of transfer RNA precursors Protocol 6 . Contmued • 20% lv •v) aqueous Triton X- 100 IBio Rad . 161 ·0407); equilibrated overnight prior to use • 0. 15 M PMSF (Sigma P 7626). prepared fresh in propan-2-ol and added fresh to buffers just pnor to use; NB Thts protease mhibitor ts roxie and should be handled With care. • Hepann-agarose (0 8- 1 mg hepann ml bed volume; B1o-Rad 153-61731 Two columns are requ1red. one containing 10 ml bed volume per litre of starting cell culture (see Protocol 5 I and one containIng 1 ml bed volume (in a disposable column or syringe) each equilibrated 1n ME buffer

• HSM buffer ( 1.0 M KCI, 0 . 1 M Tns- HCI pH 8.0, 10% glycerol, 2 mM EDTA. 5 mM spermid1ne, 1 mM DTI ', 1 mM PMSF •t • LDM buffer (20 mM Tns - HCI, pH B 0, 10% glycerol. 5 mM 2-mercaptoetha.nol 1 mM EDTA. 0 . 2 mM PMSF', 0 .02 % Triton X- 100) • ME buffer (LDM buffer but With o _g q Tnton X - 100 and 0. 1 M (NH,t 2SO,,) • ME buffer w1th 2% Tnton X -100, 0.1 M (NH.t 2SO. • Storage buffer A (20 mM Tris- HCI, pH 8 .0 . 0 2 mM EDTA. 0 . 5 mM DTI 0 . 5% Tnton X -100, 35 % glycerol)

• 0.5 M sperm1d1ne (phosphate salt, Sogma, S 0381). pH to 7. 5 With HCI; store frozen at - 20 °C

Method NB Carry out all the following procedures at 2-4 °C. 1. H1gh-salt wash ofnuclei. Resuspend the yeast nuclear pellet in 7 5 ml of HSM buffer. Collect the nucle1 by centrifugation at 1 7 000 g for 1 5 min at ..:~ °C 2. Low-detergent wash . Resuspend the pellet in 7 .5 ml of LDM buffer and incubate the suspension for 10 min on ice. Recover the membrane fraction by centrifuging at 1 50 000 g for 60 min at 4 °C. 3. Membrane extraction. Resuspend the membrane pellet in 10 ml of ME buffer containing 2% Triton X-1 00. Incubate the membranes for 30 m11'1 on ice with occasional miXing. Remove any remain1ng particulate material by centrifuging at 1 50 000 g for 60 min at 4 °C.

4. Apply the supernatant to the large heparin-agarose column. Collect 2 ml fractions throughout each of the followmg wash and elution procedures Wash the column w1th 20 ml of ME buffer

5. Elute the column stepw1se w1th 20 ml of ME buffer conta1mng 0.25 M (NHl4bS04, and then 20 ml of ME buffer plus 0.45 M (NH 4 ) 2 S0 4 • The endonuclease elutes in the latter fractions. 6. Dialyse the eluted endonuclease against 3 x 1 litre of fraction buffer for 1 h each t1me at 4 °C with stirring. 7 . Concentrate the dialysed endonuclease by applying 1t to the 1 ml hepann -agarose column. Elute the endonuclease with 2 ml of ME buffer plus 0 .45 M INH 4 l2S04. 8. Dialyse the eluate overmght at 4 ° C agamst one litre of storage buffer. Store the dialysed endonuclease solution at - 20 °C 1n 0 . 1 - 0 5 ml aliquots. • Add these reagents JUSt prior to use from stock solutions.

190

J

Chris L. Greer This procedure yields an endonuclea~e preparation which is approximately equivalent to Fraction V of ref. 35. Endonuclease activity averages 100 U/ ml where I Unit of endonuclease cleaves 20 pmol pre-tRNA substrate min t under standard reaction conditions (35). Significantly, the preparation contains only traces of contaminaung phosphatase and nuclease activities and is suitable for most studies of processing reactions including thetr substrate requirement'>. reaction products, and general reaction kinettics.

3.3 Preparation of highly-purified processing components Ideally, comprehensive biochemical characterization of a tRNA processing step should be carried om through its reconstitution from purified components. Often, thb is accomplished by extensive purification of processing enzymes from homologous cell extracts. This approach is particularly applicable to reactions requiring multiple components or complex assemblies, does not include a requirement for cloned genes or cDNAs, and allows for the inclusion of essential post-translational modifications. However, this approach can be experimentally challenging or even impracticable when the c•ell or tissue source is difficult to obtain in large quantities, or when the processing components are present at only trace levels in cells or are unstable in homologous extracts. An alternative approach is expression at high levels in a heterologous system followed by purification by conventional or .affinity methods. A prerequisite for this approach is an appropriate cloned DNA sequence encoding the desired product. Advantages include the ability to produce altered processing components in vitro by modifying the expressed gene (particularly important where the gene encodes an essential product) and the beneficial effect of efficient and high-level expression on both the degree of purification required to obtain a homogeneous product and to produce large quantities of the purified product. Disadvantages in this approach are those that favour homologous extracts: limited post-translational processing of the expressed protein factor and difficulty in reconstituting multicomponent or complex systems. Prorocol 7 provides an example of a bacterial expression system which allows the rapid and efficient punfication of .a yeast processing enzyme (RNA ligase). The bacterial expression vector used in this procedure, pDAKC (37), allows expression of yeast ligase as a translational fusion protein with bacterial dihydrofolate reductase (DHFR). This vector, derived from plasmid pKK223-3 (38), includes both the rae promoter for high-level expression on induction with isopropyl-13-D-thiogalactos.ide (IPTG), and a transcription terminator, rrnB T l/T2. The fusion protein, can be purified from bacterial cell extracts using a rapid and simple affinity method based on DHFR binding to metbotrexate-agarose (39). The affinity-purified protein can be used directly in processing studies, or contaminating nucleic acids and enzyme-bound folate can be removed using a DEAE-cellulose flow-through procedure as described in Protocol 7. 191

6: Processing of transfer RNA precursors

Protocol 7 . Purification of DHFR-Iigase fusion protein from E. co/, Equipment and reagents 47. 5mM K 2 HP04 1. 0 18M KCl, 10'1' glycerol)

• Dosposable chromatography columns (QS-Y, lsolabl

• E. coli, strain SW 1061 (pDAKC). C600 Dlon 100, hsdR , hsdM· , supE, thr- 1, leuB6, thi- 1, lacY1, IF' laci50 :: TN5); transformed woth the ligase expressoon vector, pDAKC

• M6E buffer (0. 1 M potassoum borate, pH 9.0, 1.0 M KCl, 1 mM EDTA, 2 mM folic acod, 1 mM 2-mercaptoethanoll. A del the folate and mercaptoethanol just prim to use

• LKA growth medium (8 gil NaCI, 5 gil yeast extract, 10 g/1 Bacto-tryptone (Dileo Laboratories) plus 50 "g ml each of kanamycin and ampicillin)

• MB buffer (50 mM potass1um phos phate, pH 6 .0 h.e. 44 mM KH 2 PO,. 6 mM K 2 HP0 4 ), 0 . 2 M KCl , 0 . 5 mt.-1 EDTA, 1 mM 2-mercaptoethanoll

• Lysozyme stock solution 117 mgl ml in 10 mM Tris-HCl, pH 8.0; Sigma L 6876} • Brij 58 solution (polyoxyethylene 20 cetyl ether; 50 mglml aqueous solution; Sogma P 58841 • DNase 1 solutoon 113 mg ml stock 1n 10 mM Tris-HCl, pH 8.0 ; S1gma DN-251 e 0.1 M lPTG • 0 .2 M PMSF (P 7626 Sigma), prepared fresh on propan-2-ol and added fresh to buffers just prior to use. NB Th1s protease inhibiror is toxic and should be handled with care • 0. 5 M EDTA (pH 8 .0 woth NaOHl

• Methotrexate-agarose. Equilibrate moetho trexate - agarose slurry IPoerce 20206 H in MB buffer Use 20 ml of slurry, contaon· ing 10 ml (settled volume) of moetho trexate - agarose and 10 ml of MB buffeo e DEAE -cellulose IWhatman DE-5 2. 4057-200). Equilibrate 3 ml lsnttled volume) per litre of startong cell cult•Jre in DEAE buffer • MW buffer 1200 mM potassium phO$ phate, pH 6.0 (i.e. 176 mM KH 2PO. 24 mM K 2HP0 4 1. 1 M KCl, 1 mM 2-mercaptoethanoll • FR buffer (100 mM potassium phosphate pH 6.0 ) (i.e. 5 mM KH 2P04 and 95 mM K 2HP04 l

• 1.0 M MgC12 e 40 mM Tros-HCl. pH 8.0 • Solid 1NH4 l 2 S0 4 , finely ground e DEAE buffer (50 mM potassium phosphate pH 8.0 (i.e. 2. 5 mM KH 2PO• •

• Storage buffer B ISO mM potassium phosphate, pH 6.0, 0 .1 M KCI, 5 mM 2-mercaptoethanol, 35% glycerol)

Method NB Steps 4-13 are carried out at 2 - 4 °C with prechilled equipment and buffers

Growth of bacterial cells 1. Inoculate 500 ml of LKA growth medium with bacteria and grow overnight at 37 °C with aeration until the A 600 is approximately 3 .0 . 2. Inoculate 10 litres of LKA medium with 500 ml of the overnight culture and grow at 37 °C until the A 600 is approximately 1.0 (3.5-4.0 h). 3 . Add IPTG to 0 .5 mM and continue the growth for 45 mon (A 600 should reach 1.2- 1.6).

CoMinued 192

Chris L. Greer Protocol 7. Continuc<Jd 4 . Harvest the cells by centrifugcng at 4200 g for 10 men at 4 °C Resuspend the cell pellet en 500 ml of 40 mM Tris-HCI, pH 8.0, and centrifuge at 6000 g for 1 5 min at 4 °C en a preweighed centrifuge tube. Record the weight of the cell pellet (typccally 1 8-2.8 g per litre of original culture). Store the cells at - 70 °C, they are more readily lysed (in step 6 below) after a freeze/thaw cycle. Extraction of fusion protein

5 . Add 2.14 ml of 40 mM Tris- HCI, pH 8.0, per gram of cell paste and measure the volume IV, I of the resuspended cells. Add 0.005 x V, of 0.2 M PMSF, 0.01 x V, of 0.5 M EDTA and 0.04 x V, of lysozyme stock solutcon. IncubatE! the mixture at 2-4 °C for 30 min with slow stirring. 6 . Add 0.04 x V, of Brij 58 solution. Incubate for 30 min at 2- 4 °C with slow stirnng The! viscoscty should increase wcth cell lysis 7. Add 0 082 x V, of 1 0 M MgCI2 , and 0.004 x V, of DNase I solution. and incubate for 30 rnen Highly viscous samples may require the additcon of a second aliquot of DNase I solution and further incubation for 30 men. 8 . Remove the cell debris by centnfugeng at 23 000 g for 1 h at 4 °C Carefully recover the supernatant and measure its volume. Add 0.226 g of solid (NH 4 ) 2S0 4 per ml of supernatant; make the additions in small amounts wactmg until the (NH 4 ),S0 4 has fully dissolved before further additcon 9 . Centrifuge the suspension at 12 000 g for 40 men at 4 °C. Recover the supernatant and measure cts volume. Add a further 0 212 g of solid (NH 4 ),S04 per ml of supernatant slowly with mcxcng. Centnfuge at 1 2 000 g for 40 rnin. 10. Resuspend the protein pellet in 18-20 ml of MB buffer. Measure the protem concentration by the method of Bradford (40). Adjust the final concentration to 20 mg protein/ml wcth MB buffer (typical yields are -75 mg protein 10er litre starting culture). 11 . Add 1 ml of methotrexate-agarose slurry per litre of starting culture. Place the resulting suspension in a dialysis bag and dialyse it agacnst two 4 litre batches of M B buffer for 1 2-1 6 h (mix the suspension occasionally by cnverting the dialysis bag). 12. Mcx the dcalysed sample thoroughly and transfer ct to a dcsposable chromatography column, allowcng the column to flow at -0. 5 ml/mcn. Collect 7. 5 ml fractions. 13. Wash the column at 0. 75 mi, men wcth MW buffer untcl the eluate reaches A 280 <0.5 (typccally after 20-30 column volumes) 14. Elute the bound protein with MBE buffer at 0.5 ml/mcn, collecting 1 5 ml fractions Measure the protem content (step 10) en the eluted fractcons and pool those with protein concentrations ~ 20°1i> of that of the peak fraction. Dcalyse tthe pooled fractions against 3 changes of FR buffer ( 1 litre each time) over 10-12 h.

Continued

193

6 Processing of transfer RNA precursors Protocol 7. Continued 15. Concentrate the eluted protein by prec1p1tation w1th (NH4 12SO, "S follows: measure the volume of the pooled fraction and add 0.516 g INH4 12 S0 4 per ml Collect the precipitate by centrifugmg at 12 000 g ' or 45 mm.

16. Resuspend the pellet in 5 ml of OEAE buffer and d1alyse the solut1on against 3 changes of this same buffer ( 1 litre each time over 12-15 hi.

17. Apply the dialysed solut1on to the DEAE-cellulose column. Maintain the flow rate at 1.5 ml min and collect 2 ml fractions. Wash the column With 1 column volume of DEAE buffer. collecting fractions as before. Measure the prote1n concentrations of the fractions and pool those fractions w ith values ~ 10% of the peak fraction

18. Concentrate the purified protem by precipitation With (NH 4 hS0 4 as described in step 15. 19. Resuspend the prec1p1tated protem in 1 ml of storage buffer Band dialyse 1t against 3 changes of this same buffer, 1 litre each time over 15-20 h. 20. Store the dialysed preparation 1n aliquots at -20 °C.

L_

Final preparations obtained using Protocol 7 typically contain 1-2 mg protein/ ml, corresponding to yields of 0.1-0.2 mg per litre of starting cell culture. An example of the purification of the DHFR/ Iigase fusion protein is shown in Figure 2, including the methotrexate-agarose affinity chromatography (Ftgure 2A) and SDS-PAGE analysis of pooled fractions obtained by affinity chromatography (Figure 2B). Ligase activity in the final purified fraction is typically I000-3000 U/ml, where I Unit of enzyme activity ligates I pmol tRNA substrate per min 1 (41). Other important features of the procedure include the following: (a) The peptide linker in the fusion protein includes a Factor X cleavage me which allows separation of the yeast and bacterial protein segments by endoproteinase treatment (42). However, the author has found that the acti,ity of the ligase is not affected by the presence of the DHFR segment. (b) Induction of the cell cultures with lPTG for long periods ( ~ 2 h) resu ts in the accumulation of subfragments rather than full-length fusion protein. In fact, for many deletion derivatives, recovery of the intact fusion protein is better wtth uninduced cells. Thus, a comparison of induced and uninduced cell extracts should be made before settling on a final protocol. (c) The ionic strength of the DEAE buffer in the DEAE chromatograph"' is intended to aJiow retention of contaminating folate and polynucleotides without retention of the fusion protein. Recovery in this step is sensith·e to smaJI changes in ionic strength and so the conductivity of buffer, eluant, and ~ample should be carefully monitored. 194

Chris L. Greer

A.

B. ,..

0.8

rrc;110ns Doofed

-

-<

X

97-

If)

x 0.6

X

x'

Cl

If)

X

< c G)

660.4

... 0

a..

x'

0.2

. x

~.X

45-

it,

x,

x '

0.0

·x.x'

X

X

31

' r · ·.Y. x·X

2

4

6

8 1 0 1 2 1 4 1 6 1 8 20 22 M

Fraction Number

OAKC

OAC

Figure 2. PunficatJon of DHFR-Iigase fus1on protein. (AI Elut1on profile of methotrexate-agarose affinity chromatography (Protocol 7). The protein content:> of the elution fractions were measured by the method of Bradford (40). The A 595 values shown are for 20 JJI aliquots in 1 ml assays Fract1ons 9-1 9 were pooled for subsequent processing (see Protocol B). (B) Samples of the pooled elution fractions in lA) were concentrated and thon analysed by electrophoresis in 50S polyacrylamide gels and stamed with Coomass1e blue (89). lanes contained - 10 JJ9 of an extract from cells expressing the 110 kDa DHFR -Iigase fusion protein (DAKC) or a 91 kDa deletion derivative (DAC, lacking amino ac1ds 397-595; ref 37) as indicated at the bottom of the panel. Molecular weights (kDa) of protein markers (lane Ml are indicated on the left

(d) While native gel electrophoresis to measure pre-tRNA binding could not be carried out with fractions prior to the DEAE-ceUulose step, the enzymatic activities of the methotrexate-agarose and DEAE-cellulose fractions were similar (37) and an abbreviated purification (omitting steps 16-18 of Protocol 7) is suitable for many applications.

4. Pre-tRNA processing assays 4.1 Strategy In addiuon to procedures for providing RNA substrates and active processing extracts, a third essential element in a biochemical approach to tRNA processing is an accurate, sensitive, and practical assa) method. lmponam considerations in the design and implementation of an appropriate assa) fall into two categories. These are: 195

6: Processing of transfer RNA precursors

• the choice of reaction conditions • the basis for the ana1}1ical method used to distinguish between substra es, intermediat~. and reaction products. Reac1ion conditions chosen for initial characterizations are often intended to mimic, within practical limitations, the normal cellular environment. Primary variables include pH, temperature, ionic strength, the nature of counterions, incubation times, and the concentrations of co-factors, substrate, and processing components. The effects of stabilizing agents, including spermidine and Mg~­ ions which bind to and stabilize tRNA structure (I 0), and reducing agents should also be examined. An example of a completely reconstituted splicing assay in which reaction conditions have been optimized is described in Protocol 8 (Section 4.2). The design of an analytical method for measuring reaction products depends on the nature of the processing reaction. As described in Section 4.2, tRNA processing reactions are of three types: removal of extraneous sequen.:es, addition of non-encoded bases, and introduction of base and ribose moclifications. Reactions in the first two categories result in substrates and products which differ both in size and sequence, so assay methods are generally designed to exploit these clifferences. Thus electrophoretic separation (20. 21) and differential hybridization to oligonucleotide probes (16) are commonly applied to monitor the first two types of reaction. Electrophoretic separation is incorporated in Protoco/8. Reactions in the third category, the introducuon of modifications, generally do not alter the electrophoretic mobility sufficiently to serve as the basis for an assay. Instead, indirect methods must be used , most of which rely on an analysis of the base composition of substrates and reaction products. An example of an indirect method for analysing base modifications is described in Protocol 9.

4.2 Assay of pre-tRNA splicing The presence of an intron is characteristic of a limited subset of eukaryo1 ic nuclear tRNA genes (43). Splicing of the transcripts of these genes requires \.'O distinct activities: a site-specific endonuclease and an RNA ligase. The splicmg reaction can be reconstituted in vitro using pre-tRNA substrates, prepared as described in Sections 2.3 and 2.4, and splicing enzyme fractions, prepared as describc!d in Section 3.2 and 3.3. Optimized conditions for in vitro splicing and the use of gel electrophoresis as a basis for the separation and quantification of the reaction products are illustrated in Protoco/8. Important features of this assay include the following: (a) The· yeast endonuclease preparation is a detergent extract of a membrane fraction and so detergent (Triton X-1 00) is required in this assay (in the I0 ><splicing cocktail) to maintain enzyme solubility. 196

Chris L. Greer (b) The stabiJizing effect of spermidine varies among pre-tRNA subtrates wit h a modest effect for some and a strict requirement for added spermidine for others (cf. ref. 35).

(c) Joining by RNA ligase involves two NTP-dependent reactions; a GTPdependem substrate phosphorylation and an ATP-dependent adenylylation of protein and RNA intermediates (Belford, H. G., Westaway, S. K., Abelson, J. , and Greer, C. L., in preparation). The nucleotide mixture in the assay provides both NTPs at concentrations 2-5 times their respective K app (NTP concentration required for half-maximal velocity) values. (d) The splicing reaction is optimal over a relatively narrow range of NaCl and KCl concentrations (5-50 mM). However, glutamate, a more relevant physiological anion than Cl - (45), can be used at con cent rat ions up to 0.5 M with little deleterious effect on splicing and at the same time reducing the non-specific binding of ligase by RNA (46). Thus, as has been suggested for other protein/ nucleic acid interactions, the use of glutamate as a coumerion may, under certain conditions, enhance specificity. (e) Digestion of protein components with proteinase K (in the stop mixture, see Protoco/8, step 3) prior to precipitation with ethanol improv•es recovery and enhances subsequent electrophoretic resolution of the reaction products. This is especially apparent for reactions containing concentrated or complex protein mixtures.

Protocol 8 . Pre-tRNA splicing assay Equipment and reagents •

32

P-Iabelled pre-tRNA substrate

I- 5000 d.p.m.lfmol; 5 fmoi/IJII prepared as in Protocol 3 or 4 ; each assay requires 5 fmol

• Dilution buffer (2 5 mM NaCI, 20 mM Hepes-KOH, pH 8 .0, 1 mM OTT O.SmM EOTA, 0 . 5% Triton X- 100, 20% glyceroll

• Yeast endonuclease prepared as in Protocol 6 ; each assay requires 0.1-1 .0 x 10 • units • OHFR-Iigase prepared as in Protocol 7; each assay requires 0.1-1 0 x 1o- 3 units • 1M

on (see Protocol 5 I

• 0 .25 M spermidine 1Protocol6), pH 7. 5 with HCI • Sterile water (Protocol 11

• to x

• 10 x nucleotide mixture ( 1 mM A TP and 0. 1 mM GTP m TEO 1 buffer) ; store at - 20 ° C

splictng cocktail 10.25 M NaCI, 0 .2 M Hepes - KOH, pH 7.5, 50 mM MgCI 2 , 25 mM spermidine, 10 mM on. 4 % Triton X- 100)

• Polyacrylamide gel I 12% acrylam1de, 0.4% bisacrylamide, 8 M urea. 1 xT8E buffer; 20 x 20 x 0. 1 em); see Protocol 3 • Gel sample buffers. elution buffer, 2.0 M ammonium acetate, pH 7.0 . TBE buffer, Quik-Sep columns as in Protocol 3. • Stop mixture and TE0. 1 buffer as in Protocol 4 • 2.0 M sodium acetate, pH 5.0

CorJtinued 197

6: Processing of transfer RNA precursors Protocol 8 . Continued

Method 1.

Dilute enzyme stock solutions, where necessary, using dilution buffer and leave to equilibrate for 0 . 5-1 h on ice (equilibration after dilution is essential for detergent-solubilized fractions) . Dilute endonuclease fractions to 0. 1-1 x 10 3 Units/IJI. Dilute ligase fractions to 0. 1-1 x 10 2 Unitsl i.JI.

2.

To a microcentrifuge tube on ice, add the following in the order given.

3.

• sterile water • 10 x splicing cocktail • 10 x nucleotide mixture

5 IJI 1 IJI 1 i.JI

• yeast endonuclease eDHFR-Iigase • 3 2P-Iabelled pre-tRNA substrate

1 i.JI 11JI 1 i.JI

Incubate the mixture at 30 °C for 5-20 min. Stop the reaction by adding 0.1 vol. of stop mixture. Incubate for 10 min at 50 °C.

4 .(a) For procedures requiring fine electrophoretic resolution (i.e. molecules differing by less than 1 Oo/o in chain length) Concentrate the reaction samples prior to electrophoresis by ethanol precipitation: adjust the volume to 80 i.JI with sterile water, add 20 l.ll of 2M sodium acetate, pH 5.0, 20 i.Jg of glycogen carrier (Protocol 1 ), and 0.25 ml of ethanol. Leave on crushed dry 1ce for 30 min. Recover the precipitated RNA by centrifugation at 12 000 g for 20 m1n at 4 ° C. Resuspend the dried pellet in 5 i.JI of gel sample buffer. (b) For procedures requiring limited resolution (i.e. molecules differing by more than 10% in chain length).

5.

Analyse the RNA products directly by electrophoresis by adding 1 assay volume ( 10 i.JI) of gel sample buffer. Denature the samples immediately prior to electrophoresis by incubating for 5 min at 65 °C.

6.

Load the samples onto the polyacrylamide and electrophorese as described in Protocol4 (step 7) allowing the bromophenol blue marker to reach the bottom of the gel (2.5-3 hat 20 mA) .

7.

Disassemble the electrophoresis apparatus leavmg the gel on the glass backing plate. Wrap the gel and plate in cling film and mark the sides and corners with radioactive or fluorescent ink for later alignment of gel and film .

8.

Visualize the RNA bands by autoradiography (e.g. 8- 12 h exposure at - 70 °C using Hyperfilm-MP and an intensifying screen). Develop the X-ray film according to the manufacturer's instructions. Align the gel and the autoradiograph on a light box. With a razor blade, cut out slices from the gel corresponding to the tRNA precursor and its reaction products and determine the amount of radioactivity present by measuring the Cerenkov radiation . Recover the RNA products from the gel slices by elution as described 1n Protocol 3 (steps 21-24).

9.

10.

198

1 0 ' - - - - ' - - - ' - - _ _ . J - - - ' - - - - ' ---' 3 4 5 0 2 [ GTP]

0I

05

1

25

5

(GTP)

(uM)

Figure 3 . Pre·tRNA splic1ng assay. (A) Assays (10 iJI, see Protoco/ 8) contained pre·tRNA~'~­ substrate, 10 iJM ATP, and the indicated concentration (iJM) of GTP Reactions were incubated w1th endonuclease for 10 min at 30 °C followed by the addition of ligase and further incubation for 20 min at 30 °C The products were separated by gel electrophoresis and visualized by autoradiography (Protoco/8, steps 7 and 8). The identities of labelled products and the posit1on of the xylene cyanol marker are indicated on the left as follows: pre-tRNA, pre-tRNA substrate; tRNA, spliced tRNA product; halves. exon products of cleavage by tRNA endonuclease . (B) The reaction products separated by gel electrophoresis in panel A were quantified by measuring the Cerenkov radiation in gel slices (see Protoco/8, steps 9 and 10). L1gase activity (o/o Joined) was calculated as described in ref 44

The results of a series of splicing assays using labelled pre-tRNA~' as substrate are shown in Figure 3. Panel A shows the autoradiograph of the gel. The endonuclease cleavage products (tRNA 5' and 3' exons and the intervening sequence) migrate between the dye markers in this gel system. The joined product (spliced tRNA) migrates behind the xylene cyanol marker and is well resolved from endonuclease products and from residual precursor.

4.3 Assay of base modifications and tRNA splice junctions 4.3.1 Strategy Transfer RNA biosynthesis includes the post-transcriptional introduction of a complex and extensive set of specific base and ribose modifications (reviewed 199

6: Processing of transfer RNA precursors in refs 7, 8). These modifications are essential for normal t RNA function. influencing decoding potential, translational efficiency and fidelity, and conformational stability. Reactions required to generate the full range of modifications found in a mature tRNA include simple methylation, reduct1on and isomerization reactions, as well as more complex side-chain addition and transglycosylation reactions. These reactions are catalysed by an extensive set of site-specific modifying enzymes which are thought to act at discrete stages in a tRNA processing pathway (15). The functions of these specific modifications, the substrate recognition properties and catalytic mechanisms of the modifying enzymes, and the ordering of the processing events are important areas of study in tRNA biosynthesis. Most current biochemical methods for anaiysing specific tRNA modification reactions involve the following steps: (a) Incubation of pre-tRNA substrates with proctessing extracts; (b) Isolation of the reaction products; (c) Digestion of the RNA product to its constituent nucleotides, or nucleosides; and

oligonucleotide~

(d) Separation of the modified constituents from their unmodified counterparr.. on the basis of distinct chemical or physical properties. The requirements for the first step have been discussed in Sections 2 and 3 Isolation of the reaction products may simply consist of phenol extraction followed by ethanol precipitation (see Protocol3) or more extensive purification by gel electrophoresis (see Protocol 8) or chromatography (22, 23). Isolated reaction products are then digested to their constituent oligonucleotides. nucleotides, or nucleosides by treatment with the appropriate nucleases or chemical cleavage reagents. In this step, the effe,;ts of modifications on the cleavage reaction must be considered and examples of the use of nuclease PI and snake venom phosphodiesterase are described in Section 4.3 .2. In the final step, digestion products are resolved by TLC (8, 47), HPLC (48, 49). or combined liquid chromatography/ mass spectroscopy (LC/ MS; 50, 51). The digestion products may be detected and quantified by UV absorbance when unlabelled substrates have been used or by radiometry or autoradiography for the products of labelled substrates. Labelling with ! 32P] orthophosphate m vivo (Protocol 3) and post-labelling procedures in which the RNA is labelled after synthesis (47, 52, 53) result in uniform labelling and potentially allo\\ the detection of all the modified nucleotides present in a single digestion Where the labelled RNA substrate has been synthesized in vitro, the ability 10 detect a specific modified nucleotide depends both on the choice of the labelled nucleotide and the specificity of the cleavage reagent used in the subsequent digestion (i.e. whether phosphates are retai ned in the 5' or 3' position in cleavage products). These two factors must be considered in designing procedures which include in vitro transcription. For example, transcription with 200

Chris L. Greer [ a-32 P) GTP and subsequent digestion with nuclease P l (to generate 5 mononucleotides) would be effective in detecting modifications at G residues but would not allow the detection of other modified bases. The tRNA products of the yeast splicing enzymes have a characteristic junction structure consisting of a 2 ' -phosphomonoester-3 ,5 ' -phosphodiester linkage (41 ). This linkage is resistant to most cleavage agents and therefore yields a characteristic dinucleotide upon nuclease PI digestion of spliced tRNA. However. the nucleaseresistant dinucleotide can be isolated by TLC and subsequently digested with snake venom phosphodiesterase to produce mononucleotide products. Although Protocols 9 and 10 specifically describe the analysis of splice-site junctions, these same procedures apply to the detection of modified bases. Thus methods and conditions for sample preparation, nuclease digestion, thin-layer chromatography, and quantification can be directly applied to analyses of ba~e modifications in pre-tRNA and tRNA substrates. I

1

4.3.2 Identification and quantification of splice sites Proiocol 9 describes a method for identifying the splice-site junction based on sequential digestion of spliced tRNA with nuclease PI followed by separation of the digestion products by TLC on PEl-cellulose. The nuclease-resistant dinucleotide characteristic of the splice junction is then eluted and subjected to digestion with snake venom phosphodiesterase to identify its component mononucleotides, again after TLC on PEl-cellulose. Important features of this procedure include:

(a) The use of PEl-cellulose as an effective and general means for the separation of individual digestion products; (b) Washing with ethanol to remove the solvent components from the developed PEl-cellulose plates. This is essential prior to the elution of the dinucleotide and its subsequent digestion and chromatography of the products. Washing with ethanol is effective only for LiCl-based TLC solvents. (c) The use of triethylammonium bicarbonate (TEAB) for elution of the dinucleotide from the PEl-cellulose plates. This volatile solvent can be removed by drying samples under vacuum, thus facilitating subsequent digestions and analyses. Protocol 9. Identification of splice junctions using digestion with nuclease P 1 and snake venom phosphodiesterase Equipment and reagents • PEl- cellulose thin-layer plates 120 x 20 em Cel 300 PEl plates; Macherey Nagel or Brinkman Instruments 801 503). Wash the plates in methanol then develop once

ustng water and finally dry them. Using a soft pencil, mark sample origins 1.5 em from bottom edge of each plate with at least 1 em separating individual samples

Continued 201

6: Processing of transfer RNA precursors Protocol 9 . Continued. e TLC tank (e.g. VWR ScientifiC, KT416180-0000I

• Tygon tub1ng • 32P-Iabel1ed

spliced tRNA (labelled as •n Protocols 3 or 4 : spliced and punf1ed as 1n Protocol 81

e uv light source (254 nm, e.g . F1sher Scient•f1c 11-984-201 • Cellulose powder (S1gma. C66631 • Glass m1crocapillary tubes !e.g . 1-51JI disposable p•pettes 8eckton D1ck~nson, 46141 • Sealing compound !Seal-Ease, 8eckton D1ck~nson, 1 015) • Capillary tubing ( 1 !<: 100 mm, Kimble Products 345021 • Diamond-tip scnbing pen !VWR Products, 52865-005) • Carrier RNA prepared as 1n Protocol 1 • Unlabelled nucleotide marker m•xture (prepare by addmg 10 IJQ RNase A to 250 IJQ carrier RNA 1n 250 1JI of 10 mM Tris-HCI pH 7.4, 1 mM EDTA; incubate for 2 h at 37 C. store m•xture at -20 ''C,

• Snake !Crotalus dunssus) venom phosphodiesterase (Boehnnger Mannhe•m 108 260: 2 mg ml •n 50° glycerol); store at 4 °C • Nuclease P1 from Pemc1111um cttrmum (Calbiochem 493866. 5 mg/ml1n 20 mM sodium acetate buffer pH 5.01; store at - 20 °C • 20 mM sod•um acetate pH 5 0 e SVP buffer 125 mM Tris- HCI, pH 9.5, 5 mM MgCI 21

• 1 0 M L1CI • TEAB buffer 130% tr•ethylammon•urn bicarbonate in H2 0) Adjust to pH 9.5 w•th C02 (add dry ice shavmgs or bubble CO; through from a CO! cylinder), Store at 4 °C for up to 2 weeks

Method Precrp1tate [ 32P] tRNA. labelled as in Protocol 3 or 4 and spliced and punf1ed as 1n Protocol 8, with 20 IJg of earner RNA (Protocol 11. 2. Add 0. 51JI of nuclease P1 and transfer the mixture to a glass microcapillary tube. Seal the ends of the tube w1th sealing compound and rncubate the tube for 2 h at 37 °C 3 . Scribe the ends of each microcaprllary tube just above the seal n•. compound using a diamond-tip pen and break the ends off manually Spot the sample at the marked origin on a PEl - cellulose plate . Also spot 51-19 unlabelled nucleotrde marker mrxture alongside the experimental sample lsi. Allow the plate to dry at room temperature for 5- 10 min. Place the plate with origm spots at the bottom in the chromatography tank containing 1 M LrCI to a depth of 0 5-1 em . Develop the plate until the solvent is 1-2 em from the top edge of the plate. 4. Wash the plate by soakmg it tw1ce (for 10 mrn each time) in 75% ethanol. Allow the plate to arr dry. V isualize the nucleotide markers by UV illumination (at 260 nm) and outline each spot with a soft pencil Mark the corners of the plate with radioactive or fluorescent ink for later alignment of film and plate. Wrap the plate rn plastiC cling film and visualize labelled digestion products by autoradiography 5 . Elute the nuclease-resistant digestion product corresponding to the splice junctron as follows. Draw out the tip of a capillary tube to a fine point and connect the drawn-out end to a vacuum pump using thin Tygon

Continued 20 2

Chris L. Greer Protocol 9. Continued tub1ng (include an appropriate trap to prevent fine particulates from entenng the pump). Plug the tip of the cap1llary by draw1ng up a small amount c1f cellulose powder. With a clean microspatula, scrape the area of the PEl plate conta1ning the des1red digestion product, us1ng the vacuum to suck the PEl -cellulose flakes into the capillary tube. 6. Elute the dinucleotide by forcing a small volume 120-100 ~o~ll of TEAB buHer through the tube (from the wide end of the fine end) us1ng a synnge attached via fine plastic tubing. Collect the eluate in a microcentrifuge tube and dry it under vacuum. 7 . Resuspend the residue in 10-20 IJI of water and again dry 1t under vacuum. Repeat the resuspension and drying twice more. 8 . Resuspend the dried nucleotide in 41JI of SVP buffer and add 10 IJg of carrier RNA 9. Add 1 pg of snake venom phosphodiesterase and incubate the m1xture in a sealed microcapillary tube (step 2) for 1 h at 37 °C.

10. Break the ends off the tube and spot the sample on to a PEl -cellulose plate (as 1n step 3) Develop the plates with 1.0 M LiCI and visualize markers and labelled products as described in steps 3 and 4.

An example of the application of the method described in Protocol9 is shown in Figure 4. Here pre-tRNAPhc was labelled by in vitro transcription (Protocol 4) using [o-- 3 ~P]GTP, -ATP. or -CTP. Splicing react ions were carried out using yeast endonuclease and DHFR-ligase and the spliced products were then separated by gel electrophoresis and eluted (Protocol 8). Eluted tRNA was precipitated with 20 )Jg of carrier RNA (Protocol I). Digestion with nuclease PI was followed by TLC on PEl-cellulose (Figure 4A). RNA substrates labelled with [ 3~P] GTP or [ l 2 P] ATP gave rise to the radioactive splice site dinucleotide pG~A, "hereas [ np] CTP did not. The dinucleotides were treated "ith snake venom phosphodiesterase and their mononucleotide products were separated by TLC on PEl-cellulose (Figure 48). An alternative procedure for investigation of tRNA splice junctions is described m Protocol 10. This makes use of combined R'lase A/ RNase Tl digestion of the spliced tRNA prior to nuclease PI digestion. The products of PI djgestion are then !ii~parated by two-dimensional chromatography on cellulose thin-layer plate~. The two-dimensional system dei>cribed in Protocol/0 has been exceptionally well characterized for the separation of a wide range of modified nucleotides. Ref. 23 should be consulled for an extensive catalogue of the relative mobtlities of modified nucleotides in this system. Identificauon of labelled djgestion products is facilitated by inclusion of appropriate unlabelled markers which can be visualized under UV light. 203

6: Processing of transfer RNA precursors

A.

B.

.., .. .· •pA -

,, ...

pC-

...

·.......

-pU-

•'

C.

pG~C

pGP

0

_..pG/

+

pN

SVP

+ pC

• ' pGP--...

'pG~A-'

•• 2

l

.,.·

-PA.....--

• •• ••

- NpG~CpN--

1PI

-pC-

·pG-

'pG~A-

-

3

-On 4

" ' 6

7

8

-pGP

- Ort 9

5

Figure 4. ldent1f1cat10n of splice site. !AI Pre-tRNA"''" substrate was labelled by in VI trO transcriptiOn with [ et-·''P 1GTP (lane 11. [et-32P]ATP (lanes 2. 31. or [ et ·32 P ICTP (lane 51. Substrates were Incubated m splicmg react1ons along w1th earner RNA, and the spliced tRNA products were then purified by gel electrophoresiS Samples were then d1gested W1th nuclease Pl and the d1gestion products were resolved by chromatography on PEl -cellulose thin-layer plates and VISualized by autoradiOgraphy !Protocol 91 Lane 4 contains a sample of the earner RNA alone processed 1n the same way. !B) Spots correspond•ng to the nuclease P1-resistant dinucleotide pG:A were eluted from the plate described 1n panel A and rechromatographed in this same system without further 1ncubauon (lane 61 or after secondary digest1on w1th snake venom phosphodiesterase (lanes 7 -9; corresponding to lanes 1-3. respectively. m panel AI In panels A and B the identities of labelled products are ind1cated with astensks. Unlabelled markers, visualized under UV I ght, are outlined With dots and identified at left or right by symbols lackmg aster~ sks. Sample ongms are indiCated by Ori . ICI A summary of the sequential digestion procedure; Pl and SVP refer to nuclease Pl d1gest1on and snake venom phosphodiesterase digestion. respect1ve1y

Figure 5 provides an illustration of this procedure. Here the pre-tRl\ \ l rr substrate was uniformly labelled in vivo with [ 32P] orthophosphate (Protocol 3) and then subjected to splicing as in Protoco/8. After digestion of the spli~:ed RNA with a mixture of RNase A and RNase TJ the oligonucleotide AAgA ~P containing the spliced junction was isolated by chromatography on PE !-cellulose thin-layer plates. The eluted oligonucleotide was digested with nuclease P I and the prod ucts were resolved by two-dimensional chromatography on cell ulose plates (Protocol 10).

204

Chris L. Greer

.. IIA

~A

II

pC

.,

110

•

•pi

pG

• Ori -2-

Figure 5. ldent1ficat1on of splice Site d1nculeot1de by 2-D chromatography. Spliced pre · tRNA 1 ' ' (uniformly labelled) was digested with RNase A and RNase T 1 and the oligonucleotide product fAA~At,\p) containing the spliced JUnction was isolated by chromatography on PEl-cellulose thin-layer plates !Protocol91 . ThiS eluted ohgonucleot1de was then d1gested w•th nuclease P1 and products were resolved by two·dimens•onal chromatography on a cellulose plate (Protocol 101 For details, see legend to Frgure 4. '1- , pseudourid1ne, ori, ongrn .

Protocol 10. Analysis of tRNA splice junctions using nuclease digestion and two-dimensional chromatography Equipment and reagents • Cellulose thm-layer plates (10 ... 10cm, cut from 20 x 20 em sheets; Eastman Kodak 13255); mark sample origrns w1th a soft pencil at the bottom left corner, 1 em an from the adjacent edges • Frrst-dimensional solvent f1 00 ml of isobutync ac•d 60 ml of 0. 5 M NH4 0H); store rn a fume hood • Second-d1mens1onal solvent I 1 12 ml of propan -2-ol, 46 ml of 6 .0 M HCil

• • 2P

labelled sphced tRNA . nuclease P1, unlabelled marker nucleotide mixture as in Protocol 9 .

• RNase A !bov1ne pancreauc ribonuclease !Calbiochem, 556741 5 mg /ml stock •n 10 mM Tris-HCI. pH 7.4, 1 mM EDTA • RNase T1 (Aspergillus oryzae nbc· nuclease (Calb•ochem, 556785) ), 5 Un.tsml stock •n 10 mM Tns-HC pH 7 4

Method NB The isobutyric acid used m che chromatography first-dimensional solvent is nox10us and a strong irritant, carry out seep 5 m a fume hood.

1. Prepared spliced tRNA as descnbed 1n Protocol 8, except resuspend the f1nal spliced RNA pellet (step 1Olm 81)1 of 10 mM Tns-HCI. pH 7.4. 1 mM EDTA.

2. Add 11)1 carrier RNA, 0.51)1 of 5 mg.ml RNase A, 0 51)1 of 5 U ml RNase T1 and Incubate for 2 h at 37 °C.

Continued 205

6: Processing of transfer R.\'A prec ursors Protocol 10. Continued 3 . Spot the sample on a PEl-cellulose plate, develop with 1 M LiCI and recover the Junction oligonucleotide (this oligonucleotide is the slowest m1gratong of the digestion products) as described in Protocol 9, steps 3 -7 4. Resuspend the dried oligonucleotide in 4~JI of P 1 buffer, add 0 . 5 ~JI nuclease Pl and transfer to a glass microcapillary tube for digestion as described in Protocol 9 , step 2

5 . Break the t1ps of the microcapillary tube (Protocol 9 , step 3) and apply 3-5 ~I of the sample (in 1 ~I increments w1th intermittent drymg) at the origin of the cellular plate. Develop the plate w ith the first-dimens1ona1 solvent until the front is 0. 5-1 em from the top edge of the plate. Allow the plate to dry in a fume hood overnight.

6 . Turn the plate through 90° and develop it in the second dimension with the second-dimensional solvent until the front is 0. 5 - 1 em from the top Dry the plate and visualize the marker nucleotides and labelled products as described 1n Protocol 9 (step 4). Note that guanosine and many of ts derivatives fluoresce under UV hght in propan-2-oi:HCI. Also, the presence of excess RNA ( > 20 ~g) causes guanosine to streak in the f1rst dimension, 1 . Align the plate and autoradiograph on a light box, marking the positions of radiolabelled spots with a soh pencil. Spots can then be cut out w oth sharp scissors or a razor blade, to measure radioactivity. Yields of individual dinucleot1des or modified bases can be calculated relative to the yield of one or more other bases of known yield

Acknowledgements Heather Belford and Shawn Westaway are gratefully acknowledged for helpful comments, assistance with experimental procedures, and communication of result~; prior 10 publication. This work was supported by grants from the NIH (GM-35955) and NSF (DMB-8614092). C.L.G. is an Established Investigator of the American Hean Association.

References I. ~ichols, t-1., Willi~. 1., and Soli, D. (1990). In \.1erhods tn en:;ymology, \ ol. lSI (ed. J E. Dahlberg and J . N. Abelson), p. 377 Academic Press, San D1ego. 2. H opper.,\ K (1990) In '>fethodsmen-;,ymo/og.J, \'ol. 181 led J E. Dahlberg and J . !'-.. Abelson), p. 400. A cademic Press, San Diego. 3. Schtmmel. P R., Soli. D. and Abelson, J (ed.) (1979). Transfer RNA. srructur~. propemes and recognillon. Cold Spnng Harbor Laboratory Pre-.~. Cold Srr g Harbor, N'r. 4. Soli, D. , Abelson, J . N. and Schimmel. P. R. (1979). Transfer RNA: btological aspects Cold Spring Harbor Laboratory Pre~s. Cold Spring Harbor. "l'r. 5. Spmzl, M ., Dank, N .• Nock, S., and Schon, A . (1991). Nucleic Actds Res., 19 (suppl.), 2127 6. Cedergrcn, R J.. Sankoff, D., LaRue. B., and Grosjean, H. (1981). CRCCrir. Rei'.

Biodtem., 11 , 35

206

Chris L. Greer 7. Bjork. G. R., Eric,on. J . U., Gu,taf;,on, C. E D. Hager\all. T. G., Jom...on, Y H., and Wikstrom, P. ~l. (19~>71. Annu. Rer Brochem .. 56, :~63. 8. '\1shm1Ura. S. ( 1979). In Transfer R,\'A: wructllre-. properties and recognrtinn (ed. D Soli J \belson, and P.R. Schimmel). p. 547. Cold Spring Harbor Laboratory Preh. Cold Spring Harbor, :-.lY. 9 R1ch. ,\. and RajBhandary, U. L. ( 1976). Annu. Re1. Brochem 45. 805. 10. Kim. S -H (1979). In Transfer R.\'A: Hructure, proper/le.~ and rt•cognrtion (ed. D. Soli, J Abelson, and P. R. Schimmel), p. 83. Cold Spring Harbor Laborator) Press. Cold Spring Harbor. NY II. 'lormanl>. J. and Abel~on, J. ( 1989). Annu. Re1•. Biochem . 58. 1029. 12 Deutscher. \ 1. P. (1984) CRC Crrt Re1·. Biochem .. 17. 45. 13. Deut<.cher. ~1. P. (1990) Progr 1\,ucleic Acids Res. Mol. Brol., 39, ~09. 14 \!elton. D A., DeRobertis. E. \1., and Cortese, R. (1980). \.atzme. 284, 143. 15 '\;ishikura. K. and DeRobertis. E. ~1. 11981). J. \to/. Brol, 145. 405. 16. O'Connor. J. P. and Peebles, C. L. (1991) \.fol. Cell. Bioi., II , 425 17. Hopper, A K. Banks. F .. and E,·angelides, \. (1978). Cell. 14,211 18 Knapp. G .. Beckmann. J S., Johnson. P. F., Fuhrman. S. A • and Abelson, J . (1978). Cell, 14, 221. 19. \\allace, D. M (1987).ln.'v!ethodsinen;rmology, \ol 152(ed S.l Berger and A R. Kimmel), p. B. Academic Pres,, '5an Diego. 20. Ogden, R. C. and Adams, D . A. ( 1987). In Methods in en~ymolog;, Vol. 15~ (cd. S. L. Berger and A. R Kimmell. p. 61 Academic Press. San Diego. 21. lkemura. T. (1989) In \1/ethods m en~ymology, \'ol. 180 (ed. J. E Dahlberg and J. N. Abelson), p. 14. 22. Tanner. N K. ( 1989). In Wethods in en:.ymoiOJ?J', \'ol. ISO (ed. J. E. Dahlberg and J '-J. AbeJ,on), p. 25. 23. Nishimura, S, Shindo-Okada, '-J., and Crain, P. r (1987). In Methods in en~ymology, Vol. 155 (ed R. \\u), p. 373. Academic Pres,, San Diego 24 Sherman. F, Fink. G. R.. and H1ck~. J. B (1986). ,\.let/rods rnyeast ~enetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY. 25. Ogden, R. C., Lee, ~1.-C., and Knapp, G (1984). \.ucle1c Acids Res, 12, 9367. 26. Knapp, G . (1989). In .',fethods m en~mulogy, Vol 180 (ed. J. E. Dahlberg and J N. Abelson), p. 110. Academic Press, San Diego 27. Greer, C. L, Javor. B.. and Abelson . J (1983). Cell, 33, 899. 28. Shapero, l\1. H and Greer, C. L. ( 1991). Biochemrs1ry, 30, 6465. 29. Engelke, D. R., Gege11heimer. P .• and Abelson. J. (1985). J. Brol. Chem .• 260, 1271. 30. Rothman, J. H .. Hunter. C. P, Vall~. L A., and Steven~. T. H. (1986) Proc. !Vat/ Acud. Sci. LiSA. 83, 3248. 31. Evans. C. F and Engelke, D. R (1990) In \1/ethods m en~ymology. Vol. 181 (ed. J. E Dahlberg and J. 'l, Abelson). p. 439. Academ1c Press, San Diego 32. Ph1z1cky, E.~~ , Sch,,anz, R . C., and Abehon, J. (1986) J. Bioi Chem., 261. 29"8 33 Rauhut, R.. Green, P.R., and Abelson, J. (1990) J. Bioi Chem .. 265, 18 180 34. Green. P R and Abelson, J. (1990) In Methods m en:,ymology, Vol 181 (ed. J E. Dahlberg and J. 'l. Abelson), p. 471 Academic Pre'\\. San Diego. 35. Peeble~. C. L., Gegenhe1mer, P., and Abelson. J. (1983). Cell, 32. 525. 36. DeRobertis, E. ~1.. Black, P. and N1shikura. K. (1981). Cell, 23, 89. 37. Apostol. B. L.. \\'estawa~. S. K. -\belson. J, and Greer, C l (1991). J. Bioi. Chem., 266, 7445

20 7

6: Processing of transfer RNA precursors 38. deBoer. H. A., Comstock, L. 1\., and Vassar, \I (1983! Proc. Natl..4cad. Set. £.:SA, 80. :!I 39. Baccanari, D.P., Stone, D., and Kuyper, L. (1981). J. Bioi. Chem., 256, 1738. 40. Bradford, M \1 (1976). Anal. Biochem., 118. 197 -ll. Greer. C. L. Peeble~. C. L.. Gegenheimer, P .. and Abelson, J. (1983). Cell, 32. 53i 42. '\agai, K. and Thoger~en, H. C. (1983) In \.fethods m en;,)molog;, Vol. 153 tc:d R. \\ u and L. Grossman), p. 461 . Academic Press. San Diego. 43. Perlman, P. S., Peebles, C. L., and Danaels, C. ( 1990). In lmerrenmg sequenct in e1•olution and de,•e/opmenr (ed. E. M. Stone and R. J. Schv.artz), pp. 112, Oxford Lniversity Pre''· Oxford. 44. Greer, C. L., Soli. D., and \\'illis, I. (198-). \lot. Cell. BIOI., 7, 76. 45 Leirmo, S .. Hamson, C., Cayley, D. S., Burgess, R. R., and Record, ~1. T (198i) Biochemistry, 26, 2095. 46. Apo>tol, B. L. and Greer, C. L. (1991). Nucleic ..4cids Res., 19, 1853. 4i. Kuchino, 'r , Hanyu, N., and ~t,himura, S. (1987). In .'>fethods 111 en:;;ymolog_l, Vol 155 (ed R \\ u), p. 3'"~. Academic Pre~>. San Diego. 48 Buck, M .• Connick, M .. and Ames, B. N. (1983). Anal. Btochem.. 129, I. 49. Gehrke, C. W. and Kuo, K. C., and Zumwalt, R . W. (1983). In The modified nucleoside of transfer R,\A, II (ed. P. F. Agrh, A r\. Kopper, and G. Koppe1), pp. 59, Allan R. Ll\~ Inc., '~Y . 50. Edmond>, C. G., Vestal. ~1 .. and ~lcCio~key, J. A. (1985). Suc/eic ..4ctds Res., 13, 8197. 51. Pomeranz. S. C . and McCloskey, J. A. (1990). In Methods m en~ymology, Vol. 193 (ed. J A. \lcCioskey), p. 796. Academic Pre,\, San Diego. 52. Randerath. K.• Gupta. R. C., and Randeralh. E. (1980) In \.fethods m en:..1molog;, Vol. 65 (c:J L. Gro~sman and K \loldave), p. 638. Academic Pre~s. Ne'~ l o 53. Silberklang, M., Gillum, A. t\1., and RajBhandary, U. L. (1979). In Methods ttl en;._vmology, Vol. 59 (etl. K. Moldave and L Gro~sman), p. 58. Academac Pre\\, Ne'\ Yor~ 54. Chen. J.-Y and Martin,"<. C. (1988). J. Btol. Cltem .. 263. 13 677. 55 Krupp, G, Cheraytl, B., Frende,~ay, D., Ni~h!Jka,,a, S., and Soli, D. (1986). EMRO J., 5, 1697. 56 Leontis, \1 , DaL1o, A ., Strobel, M .. and Engelke. D. (1988) Vucleic Acids Res., 16. 2537. 5". \fcCiain, \\', H, Cruerrier-Takada, C. and Altman, S. (198'). Science. 238. 527 58. Shapero, \1. H. (1990). PhD Thesis, Univers1ty of California, Irvine. 59. Reyes, V M. and Abelson, J. (1987). Anal. Biochem., 166, 90. 60. Hall, K and Sampson. J R. (1990). Vuc/eic Acids Res.. 18, 70-11. 61 Samuelss.>n, T., Boren, T., Johan~en, T .-1., and Lusug, F (1988). J. Bioi. Chem., 263, 13 692 62. Krupp, G., 1\ahle, D., Vogt, T., and Char, S. (1991). J. Mol. Btol., 217, 63" 63. Robertson, H., Altman, S .. and Smith, J. (1972). J. B10l. Chem .. 247, 5243. 64. Fournier \1. J., Webb, E., and Tang, S. (1977) BtOchemtslry. 16, 3608. 65. Chudne:r. H. K. and Deutsc:her, M. P. (1988). J. BIOI. Chem., 263, 1518. 66. Gardiner K. and Pace, N. R. ( 1980). J. Bioi. Chem .• 255. 7507 67. Hartmann, R. K.. Toschka, H. Y., and Erdmann, V. A. (199 1). J. Baclenol., 173. 2681. 68 Thompsen, L. D. and Daniels. C. J. (1988). J. Btol. Chem., 263, 17 951

208

Chris L. Greer 69. Darr. S.C., Pace, B.. and Pace, '-· R. (1990). J. B10l Chem., 265, 12 92~ ~o. ~lanam. S. and Van Tuyle, G C ( 198~). J. Bioi. Chem .. 262, 10 272. 71. Doersen, C.-J., Guerrier-Takada, C., Altman, S .• and Attardi, G. (1985). J. Bioi. Chem., 260, 5942. 72. Gruissem, \\' ., Gn!enberg, B. \I., Zurawski, G .• Prescou. D. M .. and Hallick, R. B. (1983). Cell, 35. 815. 73. Greenberg, B. M. and Hallick, R. B. (1986). Plant A/of. BIOI., 6, 89. 74. Wang, M. J., Davis, N. ~ .• and Gegenheimer, P. (1988). E.'vfBO J., 7, 1567. 75. Kikuchi, Y.. Tyc, K., Filipowicz, W.• Sanger, H. L., and Gross, H J (1982). Nucleic Acids Res., 10, 7521. 76. Gegenheimer, P., Gab1us, H.-J., Peebles, C. L., and Abelson. J. (1983). J Bioi. Chem., 258. 8365. 77. P1ck, L. and Hurwi1z, J. (1986). J. Bioi. Chern., 261, 668-t 78. Stange, N. and Be·ier, H . (1987). EMBO J., 6, 2811. 79. Frendeway. D., Dingerman, T., Cooley, L. and Soli, D. (1985). J. B10/. Chem .. 260, 449 80. Garber, R. L. and Altman. S. (1979). Cell, 11, 389. 81. Birkenmeier, E. H., Brown, D. D., and Jordan, E. (1978). Cell, 15, 1077. 82. Ogden, R. C., Beckmann, J . S., Abelson, J. N., Kang, H . S., Soli, D. and Schmidt. 0. (1979). Cell, 17. 399. 83. Gandini-Auardi, D .. \largarit, 1., and Tocchini-Valemim. G. P. 0985). E.HBO J., 4, 3289. 84. Gruissem, W., Prescou , D. M., Greenberg, B. M .. and Hallick, R. B. (1982). Cell. 30, 81. 85. Laski, F. A, Fire, A. Z .. RajBhandary, U. L, and Sharp, P. A. (1983). Proc. '\at/ Acad. SCI. USA, 79, 5813. 86. Dignam, J . D., Lebowitz, R. M., and Roeder, R. G. (1983). Nucleic Acids Res., 11 , 1475. 87. Zasloff, M, Santos, T, Romeo. P., and Rosenberg, M. (1982). J. B10/. Chem., 257, 7857. 88. Herrenknecht, K., van Tot, H., and Gross, H. J. (1988). ,\uclelc Acids Res., 16,-713. 89. Laemmli, U.K. (1970}. Nature, 227, 680.

209

Ribozymes DAVID A. S H UB, CRAIG L. PEEBLES, and ARNOLD HAMPEL

1. Introduction The discovery of ribozymes (catalytic RNA) by Cech ( I) and Altman et at. (2) led to exciting advances in nucleic acid chemistry and biology. In addition to a key role in the processing of certain RNA transcripts, evidence supports the broader involvement of catalytic RNA in the origin of life and e\olutionar} adaptation. This chapter focuses on the catal>tic properties of group lmtrons, group ll introns, hammerhead ribozymes and hairpin ribozymes.

2. Group I intron ribozymes 2.1 The self-splitcing activity of group I introns The term ribozyme w;as coined to describe the catalytic properties of the imron in the large subunit ribosomal RNA of Tetrahymena thermophita (3). It was soon noticed that this self-splicing intron also satisfied the sequence and structural criteria for a set of introns in yeast mitochondria, designated group I by Michel et at. (4). Well over 100 group I introns have now been described (see ref. 5 for a recent compilation), most of which are in mitochondrial and chloroplast genes. However, in addition to their presence in nuclear rRNA genes of eukaryotic protists, introns of this group have also been found in genes of bacteriophage (5) and bacteria (6). Group I introns occur in genes of e'er) kind, i.e. encoding r RNA, tRNA and mRNA. Although many of these introns (like their Tetrahymena exemplar) self-splice efficiently in \•itro, others require protein co-factors (7). Howe,er, since they all share a set of structural features necessary for self-splicing (5), it is likely that all group I introns also share a common RNA-based catalytic mechanism. protein co-factors merely acting to help the RNA achieve the correct conformation. In common with the splicing reactions of group II introns in organelles (see Section 3) and snRNP-assisted pre-mRNA splicing in the nucleus (Volume I, Chapter 3), group I cleavage-ligation is accomplished through a series of trans-esterifications (Figure 1 ). Unlike the other splicing pathwars. the

7: Ribozymes ,....-..

I J El

t'~

E2

~(L~H r

~ ,r--£-2--.

EI

[

£1

£2

-.,-

..Qo,, •G - CH

+9 l;

OH

~

Q

GOH

Figure 1. Spllcong pathway and related reactoons of group I ontrons. The splicing pathw.1~ is inotoated by labelled GTP (•GTPI bond1ng on a self-splicing RNA The labelled GTP attacks the 5 ' splice JUnctoon and attaches to the 5 '-end of the ontron ( 'G - thon hnel in the forst st• ·r of splicong The 3 '-0H on the !•berated 5' exon !open box-E1 l attacks the 3 ' splice JUncttnn to join to the 3' exon (open box-E2l and form the spliced exons (open box-E 1- open box-E2 The linear lntron is released With the labelled GTP at the 5 '-end and the conserved G at th 3' end (•G-thin line-Go..) Circularization by attack of the 3 ' GOH at an internal s1te releases the labeiiEld GTP as part of a small oligonucleotode ( 'G -short thon line-0 .,). After prolongr incubat•on or at somewhat elevated pH the corcle IS attacked by hydrox1de (OH I to y od a shorter linear inrron fragment (thin llne-G OH ). Likewose for the transcnpt, at high pH or n the absence of GTP, the 3 ' splice junction is subject to attack by hydroxide ion (OH l " non-productiVe side react1on that yields the 5' exon attached to the intron (E1-thin line - 0 , I and the ~~ · exon (open box-E21

attacking nucleophile in the first step of the group I mechanism is the 3 ' -hydro:..) I group of guanosine or GTP. Consequently, the excised linear intron has an extra, uncoded G residue at its 5 ' -end. This reaction can be used to identify and characterize the self-splicing property of group I introns. Some excised introns can cyclize; the 3 '-hydroxyl group artacks a phosphc· diester bond near the 5 '-end of the intron, with elimination of a 5' oligonucleoude (3). The circular intron can then reopen, leading to additional rounds ol circularization and reduction in length. This can result m a mixture of linear and circular intron products, of various sizes, on prolonged incubation. Finably, the phosphodiester bond between the final nucleotide of the intron and the first residue of the downstream exon is especiaUy sensitive to hydrolysis. For some introns this reaction (Figure I) can be sigmficant, 212

David A. Shub, Craig L. Peebles, and Arnold Hampel even under conditions that are optimal for splicing in vitro. The product'> of this hydrolysis do not go on to splice, and so thetr presence can complicate the analysis of in vitro splicing reactions.

2.2 Detection of self-splicing introns in cellular RNA The addition of GTP to the 5 -end of excised introns (Figure I) can sometimes be used to demonstrate the presence of self-splicing group I imrons in an RNA preparation. The advantage of this reaction is that it does not require any previous knowledge of mtron or exon sequences or the identity of the interrupted gene. In practice, detection of group I self-splicing introns involves incubating deproteinized RNA, extracted from the organism under study, with [0!-3 2P] GTP and analysing the products for the presence of radioactively-labelled RNAs by denaturing polyacrylamide gel electrophoresis_ The procedure is described in Protocol I .

Protocol 1 . Detection of group I self-splicing introns8 EqUipment and reagents e 5 x GTP-Iabelling buffer 10.25 M NH4 CI, 0.15 M MgCI7, 0.15 M Tns-HCI. pH 7 5. 5 mM spermid1nel

• Total RNA 15 mg /mil, tsolated from the organism under study as described in Volume I, Chapter 1, Protocols 9-11

• 1()-"'2PJGTP 13000Ci.mmoL 10mCi·mll • Materials and equ1pment for denatunng polyacrylamide gel electrophoreSIS and autoradiography (Volume I, Chapter 1, ProtocolS or Volume I. Chapter 4, Protocol 11), for RNA samples 100-600 nt in length, a 5% polyacrylamide gel with an acrylamide:bisacrylam1de ratiO of 30:0.8 IS suitable

• Stop solution (2 5 M ammomum acetate, 25 mM sodtum EDTA, pH 8.0, 300 ~g /~1 yeast RNA ) e 0. 2M OTT • RNasm IPromega 30 un1tsl mil

Method 1. In a m•crocentnfuge tube on ice, mix the following : • 5 x GTP-Iabelhng buffer

2 (JI

e0.2 M OTT

0.25 (JI

•RNasin

0.331JI

• [ a-32P I GTP

21JI

• RNA substrate

21JI •H 2 0 to 10 Ill final volume 2 . Incubate the mixture at 37 °C for 1 h and then stop the reactton by adding 150 Ill of stop solution chilled on ice and 320 IJI of ethanol cooled to -20 ° C. 3 . Leave the mixture for 30 min at - 70 °C.

Continued

213

7: Ribozymes

Protocol 1. Contmued 4. Recover the prec1p1tated RNA by centnfugat1on for 15 mm at 4 °C in a microcentrifuge. 5. Wash the RNA pellet by resuspending 1t in 1 ml of 70% ethanol and centnfug1ng as in step 4. 6 . Repeat step 5 and vacuum dry the washed RNA pellet. 7 . Analyse the RNA products by denaturing polyacrylamide gel electrophoresis. To do this, dissolve the RNA m BtJI of H 2 0. Immediately add 8 IJI of formamide-dyes loading solution and denature the RNA for 1 5 min at 65 °C Load the sampftes onto a denaturing 5% polyacrylamide gel, run the gel at 200 V overnight and autorad1ograph 1t as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 7 1. Autoradiographic exposures of up to 1-2 weeks might be necessary to detect weak signals. • Based on ref. 8.

The conditions in Protocol 1 are given as a guide and are not necessarily optimal. Many self-splicing im rons require incubation at higher temperatures (50 cc or more) and a buffer ''hose ionic strength is optimal for splicing in vitro. Also, since circularization eventually removes the labelled guanylate residue from the 5 '-end of the intron (Figure 1 ), the time course of the splicing reaction should be investigated. The concentration of Mg2 - ions should also be optimized; circle formation may require a higher concentration than splicing. Since RNA is readily degraded, precautions must be taken against contaminating RNases during all steps. Volume I, Chapter I (Section 1.1) and ref. 9 list general precautions that should be take:n when working with RNA. The choice of RNA isolation method (Volume I, Chapter I, Section 3) depends on the organism of interest. Naturally, the gentlest method consistent with good yield should be used. The presence of a labelled band on the autoradiograph is a good indication of a self-splicing intron. Some of the other possible imer'pretations can be eliminated by treating pan of the sample "ith alkaline phosphata~e before electrophoresis. Since group I splicing leaves the 32p.Jabelled phosphate exposed on the 5 '-end of the excised intron, it should be removed by the phosphatase. In addition to the excised int ron, the intermediate splicing product, consisting of the intron plus the 3 '-exon (Figure 1), can sometimes be seen as a second labelled band of lower electrophoretic mobility. More than two prominent bands may indicate multiple introns (see ref. 10 for an example). Thus, a positive result can reveal the presence of a group l intron and allow its size, and that of the downstream exon, to be calculated. However. failure to detect end-labelled RNA in this assa) cannot be considered proof of the absence of group I intron.s, since the assay can fail to detect group I introns for various reasons including: 214

Da,·id A. Shub. Craig L. Peebles. and Arnold Hampel • the requirement for cellular components to facilitate '>pi icing in t•itrv ethe low abundance of unsphced precursors in the ~:ell, because the introns are located in rare tran~cnptl> • the RNA precursors '>plice so readily m vivo that the concentration of self-splicing substrates in the RNA isolated from the organism is too lo'' for detection

2.3 Confirmation of self-splicing by in vitro transcription of

cloned DNA containing putative group I introns -\na1ysis of the products of in t·uro transcnption can provide e\idence for self-splicing of cloned putati\e introns . If self-splicing occurs efficiently in ~·itro, spliced products corresponding in size to the ligated exom and to the excised intron should be detectable in addition to the prima[) run-off transcript. For this assay, the DNA fragment containing the putative group I 1ntron has to be cloned into a vector which contains promoters for RNA polymerase adjacent to the cloning site. There are many commercially-avallable plasmids which can be used that contain multiple cloning sHes flanked on either side by promoters for SP6, T3, or T7 bacteriophage RNA po)ymerases. U~e of both the 5 and 3 ' flanking promoter~ aiiO\\\ transcription of the cloned insert in either direction. Since prematurely-termmated or partiallydegraded transcripts can complicate the analysis of RNA products arising from splicing, the in vitro transcription mixture should be run on a denaturing polyacrylamide gel and the full-length transcript isolated by elution (Volume l, Chapter I, Protocol 8 or Volume l, Chapter 4, Protocol fJ ). In addition, the authors recommend that the template should not exceed 1-2 kbp for efficient transcription into a full-length product, best reslllt~ being obtained ,.,.ith the shortest transcripts possible. Therefore, run-off transcripts should not extend into the \ector sequences. This can be ensured by truncation of the DNA template at the multiple cloning site or within the cloned msert by digestion with suitable restriction enzymes. Volume r. Chapter I (Section 2.2) and Chapter 2, this volume (Section 2.6} give additional information about in vitro transcription method~ . Protocol 2 describes the in vitro transcription and analysis of sequence~ containing group I self-splicing introns. Templates based on pBS-M 13 • (Stratagene) and isolated by the alkaline lysis mini preparation method (II) without RNase treatment are usually adequate for m l'itro transcnption. The DNA samples should be linearized by restriction digestion and then extracted with phenol and ethanol precipitated. Contamination with RNases must be avoided during the in ~·itro transcription and subsequent steps; the reaction mixture described in Protocol 2 contain:. RNasin but other precautions should abo be taken as advised in Volume l, Chapter I, Section 1.1. The GTP concentration present in the transcription reaction mL\ture is sufficient to allow self-splicing of group I mtrons. 215

7: Ribozymes

Protocol 2 . In vitro transcription of cloned group I introns

-, I

Equipment and reagents

• la-32P)UTP

(20 mCo/ml, 800 Co/mmoll

• 5 x transcnption buffer (0.2 M Tns-HCI, pH 8.0, 40 mM MgCI 2 • 10 mM spermodine 0 .25 M NaCII e o . 75 M

on

• RNasin (Promega, 30 units/ml) • 0.5 mM UTP • 10 mM stock solutoons of ATP. CTP, and GTP

• RNA polymerase (50 unots J.ll) from T3 or T7 phage depending on the nature of the vector used e DNA template (0 2 - 0. 51-'Q II-'11 contaoning the cloned sequence of interest down· stream of a T3 or T7 promoter • Materials and equopment for denaturing polyacrylamide gel electrophoresos and autoradiography (see Protocol 1 )

Method 1. Make up the following reaction m1xture (sufficient for 8 transcription reactions) at room temperature to avoid forming a precipitate: • 5 x transcnption buffer e o 75M OTT e RNasin

8 JJI 81JI 1 J.ll

e 10mMATP

1.6 1JI

e lOmM CTP

1.6 J.ll

e 10 mM GTP

1.61JI

e 0.5 mM UTP e [ a-32 P] UTP

1.6 t.JI

1.61JI •H 20 13 t.JI 2. Add 0.4 JJI of T3 or T7 RNA polymerase and then div1de the reaction mixture into 4 IJI aliquots. 3. Start each 1nd1vidual reaction by adding 1 IJI of DNA template. 4. Incubate the tubes at 37 ° C for 1 h. 5. Stop the reactions by add1ng 8 JJI of formamide-dyes loading buffer to each tube. 6 . Denature the samples by heating them at 65 °C for 1 5 min before analysing them on a denatunng polyacrylamide gel as described in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4 , Protocol 11

L---------------------------------------------------~

If imrons are vel") actively spliced during in ~·irro transcription, pn:cursor molecules may not accumulate to high levels. In such cases, the authors carry out the transcription reaction (Protocol 2) in the presence of very high concentrations of nucleoside triphosphates (typically 2 mM of each) leaving the concentration of MgCI2 unchanged. This reduces the effective Mg2 ion concentration below the level needed for splicing, while still allO\\ ing efficient transcription. 'IJaturally, some adjustments of the NTP and M·g2 + ion 216

David A. Shub, Craig L. Peebles, and Arnold Hampel

concentratiom may be required to achieve the best compromise between high rates of transcription and lO\\o levels of splicing. Since all of the molecular spec1es illustrated in Figure I rna} be among the products of a single transcription reaction, electrophoretograms can be difficult to interpret. If the size of the Linear intron is already known from the GTPlabelling assay (see Protocol I), a band of corresponding mobility should certainly be present on the autoradiograph. If a physical map of the insert is available, the insert can be truncated to different lengths using appropriate restriction enzymes. AnalySIS of the in vitro transcripts and spliced products from these modified templates can be used to deduce the position of the intron within the original insen:. No spliced products should be detected from transcripts ending \\oithin the intron. Similarly, deletions between the promoter and the intron can reveal whether any of the bands are due to premature termination of transcription. Ultimately, the nucleotide sequence of putative ligated exon species must be determined and compared to the DNA sequence in order to characterize fully the splicing event. Full characterization of the self-splicing reaction requ1res purification of the precursor transcript, generally accomplished by denaturing polyacrylamide gel electrophoresis and elution (Volume I, Chapter I, Protocol 8 or Volume I, Chapter 4, Protocol I I). Precursors may be uniformly labelled, in which case non-radioactive GTP is substituted to start the reaction (see Protocol 1). This discussion has been limited to the group I ribozyme reaction involved in R 'A processing, namely splicing. There are, however, a large number of other reactions that have been demonstrated on cleverly-engineered intron ribozymes. The interested reader should consult Cech {I 2) for a review of several of them.

3 . Group II intron ribozymes 3.1 Identification of group II introns Group II introns were first recognized as a specific class of inten ening sequences m yeast mitochondrial genes that are disunct from the group I introns (ref. 4 and reviewed in refs 12 and 13) Each group of mitochondrial introns is different from nuclear pre-mRNA introns, based on a comparison of the boundary sequences and internal structures. Group I or group II introns may be distinguished by their characteristic secondary structures and conserved sequences (13, 14) Standardized secondary structure diagrams for group I and group II introns have been reviewed recently (13, 15). These generalized structures can accommodate the many newly-identified members of each group. Furthermore, each group can be divided into subgroups according to specific structural features that are not general for the entire group. Thus these introns represent a diverse and expanding family of molecules. A detailed description of group II intron structure is gi\en in Section 3.2. 217

7: Ribozymes

S2.;

A. TRANS-ESTER l F l CAT I ON

B. HYDROLYSIS ~ 'A

'A~cri pt (GCGAG < • ) r2Z:1

1

1

H ·o·1

1

STEP 1

k ansesterification

1

~

, linear 2/3's GCGAGP W-)e r mediate 't I

s

~

STEP 2

transeosh•rification

(GCGA I~+ V/21 1, lariat i ntron spliced exons

STEP 1

hydrolysis

~

exonl

exonl ~ ~ 2/3's lariat G...-L-"-)e r mediate (GCGA I

S G.rA

transcript

(GCGAG ~

GCGAGP

t

1

STEP 2

transesterification

,+V/21

linear i ntron

I,

spliced exons

Figure 2. Self-splic1ng pathways of group II introns . The sequence used throughout is that of the group II self-splicing intron a 5; of yeast mitochondria lA) The standard self-splicing pathway is in1t1ated by rrans-estenflcation The first step is attack by the branchpoint adenosme (AI on the 5' splice junct1on to release the 5' exon (hatched box) from the 2/3's lanat intermediate composed of the 1ntron (GAGCG-thin line) and the 3' exon (open box). The second step IS attack by the 3'·0H of the 5 ' exon on the 3' splice Junction to release the spliced exons and a lanat intron. (8) An alternative self-splicing pathway is initiated by hydrolysis and is thus uncoupled from branch1ng. The first step is attack by water 1"0") on the 5' splice junction to release the 5' exon (hatched box) from the linear 213's intermediate composed of the mtron (pGAGCG-thin lme) and the 3' exon (open box). The second step is attack by the 3'-0H of the 5 ' exon on the 3' spl1ce junction to release the spliced exons and a linear mtron. The arrowheads 1nd1cate the 3' direction or a 3'-0H end

Group II introns have been identified in the mitochondrial or chloroplast genomes of fungi, plants and protists (13). Like many group 1 introns (reviewed in ref. 12), some group II introns are capable of self-splicing in vitro (16-21). For group II introns (Figure 2A ), the first step is an attack on the phosphate group at the 5 ' splice junction by the 2' -hydroxyl group of an unpatred adenosine near the 3' splice junction. This trans-esterification reaction releases the 5' exon with a free 3 ' -hydroxyl group and creates a 'lariat' intermedtate In the second step, the 3 '-hydroxyl group of exon I attacks the phosphate group at the 3 ' splice junction to create the spliced exons and release an excised intr<m lariat. By analogy with group 1 intron ribozymes (Section 2) and other transesterification reactions, group ll ribozymes are presumed to position a Mg~ + ion as an activating electrophile and to fold specifically for alignment of the reactants to promote an in-line attack on the phosphorus atom. While the primary products of the group 1I self-splicing reaction are linear spliced exons and excised lariat introns (see Section 3.4 and Figure 2), several other 218

David A. Shub, Craig L. Peebles. and Arnold Hampel

IBS21 Exon 1

Exon2

Figure 3. Schematic diagram of group II intron secondary structure. A stylized drawing after the model of ref. 13 showing the secondary structure of a group II intron as the thin line; the curved segments are identified as unpa1red 1n the secondary structure with parallel stra1ght lines indicating helical segments. The 5' exon and 3 ' exon are indicated as heavier straight lines. Sites involved in tertiary base pairing interactions are labelled and mdicated with the grey highlight bars (EBS 1, EBS2, IBS 1, 1852, a , a ', 13. 13', E.£' ). Each domain and subdomain (within domain 1) is indicated, and the other features are discussed in the text (Section 3.2) No attempt has been made to diagram the secondary structures of domains 2 and 4.

splicing-related reactions have been described (see Section 3.5). This section will focus on the various conditions that have been used to analyse splicing and other reactions of group II introns.

3.2 Group ll intron secondary structure and functional

anatomy The distinctive secondary structure of group 11 introns is usually displayed as a 'cloverleaf' consisting of six 'domains' numbered 1-6 (Figure 3 ). Each domain is bounded by 'pairing segments' that are represented as helical stems. Each domain is separated from its neighbours by shon ' joining segments'; joining segments are conserved in length and partly conserved in sequence (13). The 5 1 splice junction is apparently selected by tertiary base-pairing interactions between exon and intron sequences. There are two exon-binding sequences (EBS I and EBS2) in subdomain D of domain I; EBS I and EBS2 are complementary to intron-binding sequences (JBS I and IBS2) in the 5 1 exon (exon I). These pairings are designated IBSl :EBSJ and IBS2:EBS2 (22). 219

7: Ribozymes

While the exon sequences adjacent to the splice sites are highly ,·ariable, there are recognizable consensus sequences defined for both 5' and 3 ' termini of the intron. The 5 ' consensus is-! GUGCG- and the 3 ' consensus is -A Y l-, where l designates the splice site (13). A base-pairing contact designated E:E' involves nucleotides near the 5 ' 1ntron boundary pairing with an internal loop of subdomain C I of domain 1 (23). Based on phylogenetic co-variation, two additional long-range base-pairing contacts are predicted to occur ,.,ithin domain I, designated cx:cx and ~:W (22, 24). Because of its complex internal structure and demonstrated role in the spectl t~ recognition of exon 1 (22), domain 1 is generally believed to contribute much to the catalytic core of group II introns. Domain 2 is rather variable in size and sequence and has no known specific role in group II activity. Domain 3 is more uniform in size, but, like domain 2, has not yet been assigned an · specific function. Domain 4 is extremely 'ariable in size and sequence, contains a large open reading frame in some group II imrons, and may be entirely deletea without severe consequences for self-splicing activity (25). Domain 5 is the mos highly conserved in both size and sequence of any domain; the presence o domain 5 may be considered diagnostic for group II imrons (13). Domain " is critical for function in vitro, playing an essential role in the first transesterification step of splicing (25), although the details of domain 5 interaction with other parts of the intron are not yet characterized . Domain 6 contains the unpaired adenosine that is the site of branch formation ( 17) and consequently may be considered one of the substrates of the ribozyme. Deletion or alteration of domain 6 can prevent branch formation, but splicing still proceeds in vitro (26-28) .. So far, there are no extensive base-pairing interactions defined between the intron and the 3 1 ex on (exon 2). However, the nucleotide on the 5 1 side and adjacent to EBSl pairs with the first nucleotide of exon 2 (28). This interaction has been designated the ' internal guide' (ig:ig' ). The last base of the intron participates in a solitary basepair with a base in J3 ; this contact is termed ,.:;' and affects the rate of the second trans-esterification step of splicing (23). In addition to ,:,· , domam 6 is needed to promote accurate and efficient reaction at the 3 1 splice junction (28).

3.3 Pl[)tential of group IT intron ribozymes for practical use Group ll introns are capable of a variety of potentially useful reaction<;. However. this potential is largely unrealized because of the hmited availabllr v. large size and slo\\ reaction rates of these ribozymes. The splicing pathwa~ resembles group I intron ribozymes but is less convenient to use since group II tntrons do not depend on a small molecule sub!>trate to initiate the reactio n Reactions of group I introns have the distinct advantage that their cleavage products can be labelled specifically by the addition of GTP (see Protocol I) Nevertheless, group II introns offer high specificity, and research is continuing for improvement of group II introns as nbozyme reagents for RNA cleavage 220

David A. Shub. Craig L. Peebles. and Arnold Hampel

and recombination. At present, there are no published reports for applications of group II introns as ribozyme agents for down regulation of gene expression

3.4 Self-splicing of group II introns 3.4.1 Introduction Some group ll introns are capable of self-splicing m vuro ( 16-21 ). The earliest examples were a5r- and bl, both of which are derived from the mitochondrial genome of the yeast Saccharomyces cerevisiae (16-18) and are members of subgroup llB (13). More recently, examples of subgroup IIA imrons, including al and a2 from yeast, have been shown to be self-splicing under some conditions in virro ( 19-21 and P. S. Perlman, personal communication). Most studies of mutants and alternative reactions have focused on as.., and bl. AU group II introns are candidates for self-splicing, but it should be pointed out that selfsplicing has not been demonstrated for every group 11 intron tested. Several protocols are currently in use by various laboratories studying group 11 introns. The original reaction conditions were devised for the prototype of self-splicing group II introns, a51' of yeast (16). The splicing conditions were similar to those for in vitro transcription of the RNA substrate, since spliced products were detected among the transcription products. Subsequent!>, modifications were made to improve the rate and yield of the ribozyme reactions. The following sections (Sections 3.4.2-3.4.4) describe the synthesis of ribozyme substrates, tht! basic procedure for studying self-splicing of group I I imrons, and variations on this basic method. Later sections (3.5.1-3.5.3) discuss other reactions of group II imrons. 3.4.2 Preparation of transcripts for self-splicing In order to detect self-splicing of a knO\.,n or suspected group II intron, it is advantageous to prepare reasonable amounts of homogeneous transcripts spanning the intron and including both exon I and exon 2 segments. This may be conveniently accomplished by cloning a restriction fragment or an appropriate PCR-amplified DNA segment containing the imron into the multiple cloning site of a plasmid with a promoter derived from SP6, T3, or T7 bacteriophage. At least 50 nt of the natural exon 1 sequence should be included to ensure that the entire length of IBS 1 and IBS2 are included. No more than a fe,, bases of exon 2 are needed. Additional sequences derived from the plasmid can be retained at either end, usually without posing problems m the subsequent transcription or self-splicing reaction. It is frequently convenient to have the total length of the exons similar in size to the intron; then the spliced exons, separate exons, and intron products are easily displayed on a single gel. To prepare the template for in vitro transcription, purified plasmid DNA must be cut at a restriction site downstream from the inserted fragment to define the 3' -end of the transcript. Restriction enzymes that leave blunt ends or 5 e:-.temions are preferred to those leaving 3' extens1ons. Cleavage withm 221

7: Ribozymes

the mtron will prevent splicing, but some intron fragments retam ribozyme activit}'. Care must be taken to eliminate traces of RNase typically used n plasmid preparations. An alternative method of preparing transcriptto 1 templates is to use PCR to amplify the exon-intron s.equence with an appropria:e promoter sequence built into the upstream primer. In vitro transcripuon is carried out as described in Volume I, Chapter I, Protocol 4. A procedure is also given in Protocol 2 of this chapter. Becau!>e yeast group II introns contain 40% uridylate residues, it is most convenient to label transcripts using [ a-32P) UTP . However, any suitably labelled nucleottde containing 3H, 14C a-35 S, or a -32 P should be successful. Since group II intron transcripts are typically over I kb in length, transcription conditions must be optimized with respect to the yield of homogeneous full-length products. Good quality RNA polymerase (United States Biochemicals) and ribonucleoside triphosphates (Pharmacia) seem to be important in this respe~t. Otherwise, full-length transcripts must be purified b:y gel electrophore~is . This may be done using 40Jo polyacrylamide gels with 0.1 OJo SDS and ~.\I urea run with TBE buffer as described in Volume I, Chapter I, Protoco/8 or Volume I, Chapter 4, Protocol 11. After the product bands ha\e been identified by autoradiography or UV shadowing, the RNA can be eluted from the gel by soaking overnight in 0.5 M ammonium acetate, 5 mM EDTA. However, bener yields and more rapid recovery are obtamed by electroeluuon in either Tris-acetate-EDTA or TBE buffers with a Schleicher and Schuell Elutrap device. After transcription, either with or without gel purification, the RNA is recovered by ethanol precipitation and dissolved in water at a concentration of I03 -104 c.p.m./IJI. The transcripts should be stored

at -20 oc.

3.4.3 The self-splicing reaction The basic procedure for self-splicing of group II introns is described in Protocol 3. It is convenient to set up the splicing reaction by adding one volume of labelled transcript to an equal volume of 2 x sphcmg reaction buffer. For intramolecular splicing assays, the concentration of the transcript is not crucial; the amount of RNA used is determined by its specific radioacti\'it) and the amount of radioactivity needed to produce an acceptable autoradiographic exposure time. The aULhors routine!} use J()C 105 c.p.m. of the transcript in a I0 IJI reaction volume. Larger or smaller reaction volumes rna} be used a~ dictated by the particular circumstances. The incubation temperature is important, since· the temperature profile is rather abrupt for self-splicing by all known group II introns (for example, ~ee ref. 16). The optimum temperature in vitro is typically 40-50 °C, with the best temperature depending on the intron. Incubation times are typically 0.5-4 h at 45 oc. When investigating the self-splicing of a group II intron for the first time, the optimal incubation temperature and reaction time must be determined in pilot experiments. 222

David A. Shub, Craig L. Peebles, and Arnold Hampel The products of the self-splicing reactton are analy~ed b> electrophoresi~ on a 40!o polyacrylamide gel "ith 0.1 cro SDS and 8 "\1 urea run in I x TBE buffer. Under these conditions, linear R~As of 0.1-2.0 kb can be resolved reasonably well. Typical runnmg times for a 30 em gel are 2-4 h at 700 V. For the large)t group II introns, 3'ro or 3.51ro polyacf)lamide gels may be more appropriate. Non-hnear RNAs, such as the lariat excised intron. display great!} retarded mobility compared to the linear form of the same total length. Consequently, the product most characteri~tic and clearly visible after ~uccessful self-splicing is the lariat intron product, since it often mtgrates more slowly thalli the starting transcript. However, the relattve mobility of the lariat varies somewhat with the degree of cross-linking of the gel matrix, the buffer salt concentration. the denaturant concentration, and the gel running temperature. Protocol 3. Basic self-splicing reaction for group II introns Equipment and reagents • :..
• 4% polyacrylamide ge• 130cm long) contaon•ng 0 .1% SDS 8 M urea and 1 I( TBE buffer plus electrophoresos and autoradiography equipment as descnbed in Volume I. Chapter 1, Protocol 8 or Volume I, Chapter 4. Protocol 11 • Urea-dye mixture 10.1 o/o br
Method 1 . In a microcentnfuge tube on ice, mix 5 of

32 P-Iabelled

~~

of 2 x splicing buffer and 5

~I

RNA.

2. Incubate the m1xture at 40-50 °C for 0.5-4 h 0 • 3 . Stop the react1on by addmg 1 IJ) of 0. 2 M EDTA to chelate the Mg 2 • ions m the reaction mixture 4 . Heat the mixture at 1 00 °C for 1 mm. and then chill it in ice Vllater. 5 . Add 1 vol. of urea-dye m1xture and load the sample on a 4% polyacrylamode/ urea gel. 6 . Run the gel for 2-4 h at 700 V using 1 x TBE buffer and then auto· radiograph the gel as descnbed in Volume I, Chapter 1, Protocol 8 or Volume I, Chapter 4, Protocol 11. • The composition of the splicing buffer can be varied as descr~bed in Sectoon 3.4 4 The condotion described here os the low-salt react1on buffer !see Table 11. • The precose temperature and time of 1ncubat1on must be determined for each intron based on pilot exper~ments.

22 3

7: Ribozrmes Table 1 . Reaction buffers for group ll1ntron self-splicing and alternative react1ons• Designationb

Tris-HCI (pH 7.5)c (mM)

MgCI2 (mM)

Spermidine (mM)

10 100 100 100

2

High ammomum chlonde

40 50 50 50

High ammomum sulphate

50

100

Low salt High Mg 2 High KCI

Neutral salt

d

0.5 M KCI 0. 5 M ammon1ur11 chloride

0 .5 M ammon1um sulphate

• • ' "

Based on ref. 29. Fmal composition 1n nbozyme reaction. Other buffers and different concentrations work as well . Greater than 50 mM mh1b1ts Significantly

3.4.4 Modification of the reaction conditions The self-splicing reaction may be accelerated by increasing the concentr:uion of Mg2 - ions in the splicing buffer (see Protocol 3) to 0.1 M as in the high Mg1 • buffer (see Table /). Ribozyme reaction times are then typically I 0-60 min. The additional inclusion of 0.5 M KCI or 0.5 M ammonium sulphate, as in the high KCI and high ammonium sulphate buffers (see Table I), further increases the reaction rate so incubation times of 2-20 min may be suffic.ient. Some group II introns will splice only under these high-salt conditions ( 19-21 ), but one should realize that the use of these alternative buffers may also activate alternative reaction pathways (see Section 3.5 below). Even higher salt concentrations can be used (29) and may provide an advantage for analysing the activities of mutant introns (30). Although concentrations of Mg1 ions and neutral salt higher than tho e listed in Table I are tolerated by aS1 intron of yeast, they result in no further increase 10 the reaction rate. Some neutral salts, such as 0.5 M liCI, sho'' little rate enhancement or change in product pattern. The anion added with the buffer or Mg2 • ions has only a relatively minor effect unless extremely high concentrations are used. Some workers use 60 mM Mg2 - ions with the same anion as the neutral salt and also include spermidine in their high-salt buffers (for example, ref. 31). Spermidine is tolerated under these conditions, but can be omitted with any of the high-salt buffers (see Table I). Addition of 0.1 a:o SDS is tolerated in all but the low-salt conditions (see Table 1 and Protocol 3 ).

3.5 Other reactions of group ll intron ribozymes 3.5.1 Introduction In addition to self-splicing, group 11 intron ribozymes also carry out a variet\ of splicing-related hydrolysis and trans-esterification reactions. The relative 2 24

David A. Shub, Craig L. Peebles. and Arnold Hampel contribution of these alternative reactions to the final pattern of self-splicing products is dependent on the ionic composition of the reaction buffer and on the nucleotide sequence of the RNA, particularly IBS I, 1852, EBS I, and EBS2 (Section 3.2 and Figure 3). For instance, the high KCI buffer (see Table I) activates additional reaction path'' ays. The high ammonium chloride buffer (see Table I) ytelds a pattern of a51 products intermediate bet\\ee-n those due to the high KCI and high ammonium sulphate buffers. The first of these alternative reactions is cis splice junction hydrolysis or SJH (see Figure 4A ). Transcripts extending from exon I through int ron domain 5 will efficiently hydrolyse the 5 ' splice junction to release exon I with a 3 '-hydroxyl group and a linear intron fragment \Vith a 5 -phosphate group (32, 33). SJH proceeds most rapidly in the presence of htgh KCI buffers (see Table 1 ). In addition, the linear intron fragment undergoes hydrolysis just 3 ' to alternative sites that are similar to IBS I. Since this reaction represents a relaxed specificity cleavage analogous to SJH, it may be termed SJH* (34, 35) (see Figure 48, C). lntron RNAs will also carry out trans SJH of substrate tramcripts contaimng the 5 splice junction (36) (see Figure 4D). Presumably in this trans reaction the group II intron is acting as a true cataly!>t, although thh. has yet to be demonstrated conclusively. The specificit> of these hydroly~;is reactions is defined largely by EBS I, a sequence block of about si\. bases. Amazing!~. imron RNA~ will also catalyse hydrolysis of the ~pliced exon product (34). This reaction has been designated 'spliced exon reopening' or 'SER' (see Figure 4£) to distinguish it from the similar SJH reaction. There are sevl"ral indications that SER and SJH are different events. For example, the group IIA imron a I of yeast carries out both SJH and self-splicing, but is inactive for SER (21). Similarly, deletion of domain 3 from a5"1 results in substantial reduction of the SER reaction while self-splicing and SJ H are much less affected (31 ). In applications of the group II ribozyme, the SJH activity acting in trans rna} be the most useful, since maximal cleavage specificity is highly predictable based on the sequence of EBSI . The role of EBS2 is not well unde·rstood, but it evidently contributes to effictent hydrolysis in trans (36). The IBSI and IBS2 target sites may be dtrectly adjacent or separated by a fe,, nucleoudes. However, only a few different sequences are available at present on which to base these predictions.

3.5.2 Splice junction hydrolysis and spliced exon reopening reactions RNA substrates for SJH and SER may be synthesized by m vitro transcription of an appropriate DNA template in the presence of [cx-lZP]UTP (see Protocol2 and Section 3.4.2). For example, exon I with domains 1-5 (El: 12345) "ill undergo SJ H efficiently (33). lntron lariat and spliced exon products are needed for SER (34). For the ribozyme reaction, mix the transcript with an appropriate high-salt reaction mixture, such as high KCl (see Table 1). For SJ H with tlhe E I: 12345 transcript, the RNA concentration is not important. Incubate the reaction for 225

7: Ribozymes

A . SJH (cis)

C. SJH* ( example 2)

~ transcri pt 'l!tith OS

1

(GCGAG ~ H-o-'~1

hydr olysis

1

H-o·~l

~+ ~CGAGP ~ exon 1

1

i ntron fmgment

B. SJH*

QG 'A~

~ GCGtl '--+-~ 1 1ntron - exon 2 t xt H-o·~l

__ll ' AS.

+

~

1

H.0.H

c+

G 'A=> GCGA P ~

't

1 -J

i ntron

+

~

GCGAG ~ (

fragment

hydrc1lys is exon 1

~

exon 2 f r agment

~~ 3 ' tntron

exon 1 pG.. tx t hydrolysis

+

.As

0. SJH (t rans)

1 ft

+

GCGAGP ~ 5' i ntron fragment

f--,

( exampl e 1)

hydrolysis

1

pG-+ fregment

E. SER ( trans)

I

V//1

tnt ron -exon 2 fragment

I I+ H.o.H

A s

spliced exons hydrolysis

j

(

GCGAG.r~ 1

lari at i nt r on

~+CJ...

exon 1

exon 2

Figure 4. Sphcmg-related ribo1•yme react1ons of group II mtrons. The exon and intron segments are represented by the same conven tions as used m Figure 2: the hatched box represents exon 1, the GAGCG-thm line 1s the mtron, the open box is exon 2. and the arrowheads 11re the 3'-0H end or the 3' direc:t10n. (AI Splice junction hydrolysis (SJH (cis!] is outl1ned tor a transcript extending through domain 5. (6) Splice junction hydrolysis occurs With a relaxed spec1fic1ty [SJH • (example 1)] at a non-canonical site (arrow) in the 3' exon using a transc11pt extendmg through the intron and 3' exon (intron-exon 2 txt). (Cl Splice junction hydrolysiS also occurs With a relaxed specificity [SJH • (example 21 J at a non·canonical site (arrowl 1n the intron usmg purified linear intron. (0) Splice JUnction hydrolysis [SJH (rrans)] is shown for an intron transcnpt extendmg through domam 5 that acts on a second transcript conta1n1ng the 5' splice junction (exon 1 txt). (EJ Spliced exon reopening [SER (trans)) is shown for an intron lanat actmg on thll purified spliced exons.

226

Da,•id A. Shub. Craig L. Peebles, and Arnold Hampel

A . TRANS- SPLICING

B. TRANS-SPLICING

( example 1) ~ exo n 1 pG-+ txt

+

( example 2)

+'A~

H.0 .H

__..0_.

:.:?

STEP 1 QG 'AS . hydro- (GCG~ ~ lysts ....-:---+•-----:,-' 1ntron - exon 2 txt

1

1

__..0_.

exon 1

~ -AS

(-GCG~ fi~ ,_J

+

1ntron - exon 2 txt

STEP 1

tr ansesterification

e~~A~

pG-+ fragment

(GCGAG-"~

Y-intron intermed1ate

1

STEP 2

1

tr ansestt'nficatlon

V/ZJ +

r?Lzr==J,

I,

sph ced exons

STEP 2

transesterification

Q 'A~

( GCGAG

s pl'i ced exo ns

GCGAG-"~

+

~

As

(

linear' i ntron

- 1·

_;

Y- intron

Figure 5. Trans-splicing react1ons of group II 1nrons. The exon and intron segments are represented by the same conventions as used in Figure 2: the hatched box represents the 5' exon (exon 1). the GAGCG-th1n lme is the intron, the open box IS the 3' exon (axon 2). and the arrowheads are the 3 '-0H end or the 3 direct•on. (A) One example of rrans-splicmg requ~res an axon 1 transcript and an 1ntron-exon 2 transcript. (B) A second example of trans-splictng requires one transcript extending through doma•n 3 (5' txt) and another transcnpt containing domams 5 and 6 and exon 2 :3 txt)

10-30 min . For efficient SER, each transcript should be used at a concentration of about 0. 1 ~ M and incubated for 30-120 min in the high KCl buffer (see Table 1 ). After the ribozyme reaction, purify the products by electrophoresis and autoradiography as described in Protocol 3.

3.5.3 Trans-splicing reactions of group

n

introns

Some unusual group n introns ha\.e been identified that are split mto two or more transcripts (37 -40). These separate RNAs presumably associate to form the correct ly-folded acti"e intron and undergo splicing in trans (see Figure 5A, B). In vitro models of such trans-splicing reactions have been constructed and used to analyse the requirements fo r efficient binding and catalysis. The first such model sy,tem supplied exon I as a transcript with a shon J extension to a second 227

7: Riboz~·mes transcript that contained the entire intron and exon 2 sequences (36). These tramcripts associated through lBSI :EBSI and IBS2:EBS2 to allow SJH and step 2 of splicing. No branching was detected. The major products \vere spliced exons, a short linear mtron fragment and a full-length linear intron. Producm : reactions occur under several buffer conditions, but with a requirement for high RNA concentrations such as 0.1 JJM for each participating molecule. This is reasonable for a second order system limited by RNA: RNA association. Later, trans-splicing systems were established between transcripts that each contained some of the intron domains and one exon (25). In effect, the self-splic 1g transcript was divided v. ithin a domain or between two domams. As befor· the most efficient reactions were observed at high concentrations of each Rl\ \ in the range of 0.1-1.0 JJ~l for each half-molecule. This style of trans-splicir .7 occurs best in the high KCI or high ammonium sulphate reaction buffers (~ee Table I) . The products are spliced exons and a Y-shaped excised intron; some separate exons and linear intron halves are also produced as byproducts. The'e trans-splicing systems have been useful for examining the roles of individual domains in the overall reactivity of the intron. For example. this approach \\ as used to show that domain 4 is entirely dispensable, while domain 5 IS essemial for splicing (25). The trans-splicing reaction is carried out using a procedure similar to that for self-splicing (see Protocol 3). First, prepare transcripts for the two halve~ of the intron, for example exon 1 with domains 1-3 (E I: 123) and domains 5-6 w1th exon 2 (56:E2). Use in ~·irro transcription of appropriate DNA templates (SecLon 3.4.2). Purification of the transcripts by electrophoresis may not be necessary. The rate and extent of the trans-splicing reaction depend on the concentrations of both reactants. Each should be present at a high concentration (at least 0.1 JJM). Small reaction volumes are useful to achieve these high concentrations: use a 4 JJl reaction volume overlaid with mineral oil to prevent evaporation. L ~e an appropriate high-salt buffer (see Table 1) and incubate the reaction mLxture for 30-120 min at 45 °C. Analyse the yield of products by electrophoresis and autoradiograph} (see Protocol 3).

3.5.4 Reverse splicing and transposition reactions The total number of phosphodiester bonds is comerved during the group II self-splicmg reaction. Therefore, each step and the entire reaction should be readily reversible. Recently, reverse reactions, where the intron lariat insert~ at the product splice junction, have been described and analysed in some detail (41, 42). Figure 6 illustrates examples of reverse splicing. imprecise 'rever~nr events have also been discovered, where the intron lariat inserts at sites related tO IBSI found within an otben\ise unrelated transcript (31, 42). Such insertion products can be 'preserved' b) reverse transcription and eDNA clonin.5. Transcripts of the resulting clones are then capable of fonvard splicinE (31 ). The reverse reactions have been termed RNA recombination or RN \ transposition (31, 42). 228

D01·id A Shub. Craig L. Peebles. and Arnold Hampel A. REVERSE SPLICING

c.rA~

(GCGA

I ~+V/21

leriet 1ntron

1

B. BRANCH ADDITION

cA~~

(GCGA 1 ~+~

I

spliced exons

1eriet i ntron

REVERSE

1

STEP 2

exon 1

REVERSE STEP 1

trans.sttnf"1Cation

transu"t•rifieation

___D_.

exon 1

~ ~ 21.3·; hriet

GCGAG.r~ermediete

(

I

1

REVERSE STEP 1

tr anststtrif1eation

C. PARTIAL REVERSAL

W 'A~ngth GCGAG ~ecursor (

GCGAGP

\

I

'A~ <--+--:.

1-J+V/21

1 ntron

1

I

spliced exons

REVERSE STEP 2

tr anststtnfieat1on

li neer 2/3's

Figure 6. Group II ontron reverse splic1ng reactions . The exon and ontron segments are represented by the same conventions as used in Figure 2 ; the hatched box represents exon 1, the GAGCG -th1n line IS the ontron, the open box is exon 2, and the arrowheads are the 3 '-0H end or the 3 ' d1rect1on . (A) Lariat mtron and spliced exons are Incubated together for complete reverse splicmg. (81 A lanat 1ntron reacts w1th the exon 1 molecule to reverse step 1 of splic1ng only . (Cl Partial reversal with a hnear 1ntron releases a free exon 1 and a hnear mtron-exon 2 (linear 2 3's).

Again, reverse splicing and transposition reactions are followed using variations on the basic splicing procedure described in Section 3.4.3 and Protocol 3. To prepare the RNA transcripts, carr) out a self-splicing reaction and purify the lariat intron and target transcript (spliced exons) by preparative gel electrophoresis and electroelution as described in Protocol 3. Purification of target transcripts may not be necessary. The substrates may also be prepared by in vitro transcription of appropnate eDNA clones. As for the other trans reactions, reverse splicing or transposition reactions require high RNA concentrations, up to 1 ~M for each RNA reactant. For an 229

7: Ribozrmes

RNA of about 300 nt, I 1-1M is equivalent to I IJ& in 10 IJI. After preparation, determine the yield and concentration of each RNA b) UV ab orption o · by calculation based on the specific radioactivity (see Volume I, Chapter I, Section 2.3.4). Set up the reaction mixture using the high ammonium sulphate buffer (see Table 1 ). Incubate the reaction mixture either at 45 °C for 30-60 min or at 30 ° C for 1-4 h, depending on the intron in use. There is evidence tha intron bl accumulates reversal products to a greater extent at 30 (42). After incubation, analyse the products by gel electrophoresis and autoradiograph\ (see Protocol 3). Reversal and transposition products are readily detected due to their reduced mobility compared to the input RNAs, despite the typica ll y low efficienc) (0.1-2.00Jo) of these reactions. There is no requirement for ei ther spermidine or elevated concentrations of Mg~ - ions to detect reverse splicing reactions although these additions may improve efficiency. lntron b l reversal product~ appear to accumulate more readil} than a5"'1 reversal products. However, for transposition it may be necessary to use elevated concentrations of up to 0.24 M \1gS0 4 •

oc

4. Hammerhead and hairpin ribozymes 4.1 Introduction Ribozymes of the hammerhead type (43, 44) and hairpin type (45-47) a ~ the catalytic centres of certain small plant vi roid and satellite RNAs (48) The full-length RNAs which contain the catalytic centre are typically 350-400 nt in length, a nd the RNA has no coding function (49). It has been suggested that the inherent catalytic activity in these RNAs is required as part of their replication cycle (50). The catalytic centre itself is the subject of t his section. The hammerhead and hairpin ribozymes form characteristic but different two-dimensional structures with their RNA substrate~. Figure 7 shows he models for the trans cleavage of target substrates b) these two classes :>f ribozyme. The hammerhead structure contains three helices (1, 11, and lll). Two of these (I and Ill) are of variable length (fi\e base pairs are sho n in Figure 7) and mvohe two regions of the Rl\A substrate on either s de of the eventual cleavage point. The third helix (II) is formed internall) and delimits the ribozyme catalytic domain of 32 nt. Thirteen of the nucleotide, involved in the structure are conserved as shown in Ftgure 7. The hairpin ribozyme has four separate helices; helices I and 2 mvolve the RNA substrat~ while helices 3 and 4 are internal. When helix I is 6 bp long, the minimum sequence of the catalytic component is 50 nt long. Both of these ribozymes cleave the RNA substrate to give a 5 1 cleavage fragment with a 2 1 , 3 ' cyclil. phosphate at the 3 ' terminus and a 3 cleavage fragment with a 5' -hydroxyl group at the 5 terminus (48). I

I

230

David A Shub, Craig L. Peebles , and Arnold Hampel Hammerhead 3'

Ill

Rlb~~~e

5

N

N • N

(9: ~

(_ :~~~'--.::..::=~~~::

Hairpin

~ U

Ribozyme

U

u

UAUAUUA GUGG CCUGG

...

....

CAC A G

s

cl

Substrate

NNNSN

CNNNNNN· • ·N

S
GO

Hellx4

3

......

A NNNNNN • • • N s

A GACC A NNNS A CAAAG

5

AG Helix 3

Hollx2

Helix 1

Figure 7 Hammerhead and ha"p1n ribozymes . The diagrams show consensus sequences of the hammerhead and ha1rp1n nbozymes. In each case. cleavage of the substrate RNA occurs at the arrow See the text for a detailed descnpt1on.

Substrate requirements differ for the t\\0 types of ribozyme The hammerhead substrate has a GUX reqwrement (helix lii) with cleavage occurring most efficiently after GUC, GUU, or GUA (51). While it appears at first that the hairpin substrate requirement is similar, in that cleavage is most efficient for substrates containing a GUC sequence, cleavage occurs before the G rather than afrer the three base sequence as with the hammerhead. In selecting the cleavage sites for the hairpin ribozyme, in addition to the GUC requirement, the adjacent base pair in helix 2 must be either a G or a C. In Figure 7, the symbol S is used for either G or C at this position. Thus the target sequence is SNGUC, with cleavage occurring between the N and G (52). Mechanistic studies on 231

7: Ribozymes

hammerhead and hairpin ribozymes represent a ne'' and exciting aspect )f biochemistry. While a limited amount of information has been obtained to d ~te (53, 54), the specific details of the catalytic mechanisms for ribozymes have ot yet been elucidated. However, because of their small sizes, the hammerhead a 1d hairpin systems have distinct advantages for study over other larger and more complex ribozymes. Hopefully, in the very near future, the role~ of specific functional groups and other features of the ribozymc!s in the catalytic mechani5m will be known.

4.2 Applications of hammerhead and hairpin ribozymes in molecular biology To date, hairpin and hammerhead ribozymes have two major applications. These are gene targeting and the generation of defined 5' or 3' ternum on RNA transcripts. i. Gene targeting Since the target RNA, or at least the substrate n:cognition sequence, is very short, hammerhead and hairpin riboz:tmes have the potential to be used a~ catalytic antisense molecules for the targeted down-regulation of specific gene expression (46, 55-58: ~ee Section 4 3).

ii. Generation of defined transcript termini Cis-cleaving autocatalytic casseues can be used to gc~nerate RNA transcnpts \\ith defined 5' and 3' termini (59-62; Section 4.4). AJPplications of this capability have been to produce plant viral transcripts with defined 3 ' termini (59) and more recently as a method to produce fully catalytically active ribozymes '·ith both a short defined 5' and 3' terminus (62). This method was then adapted to deli,er an Hl\'-1 specific hairpin ribozyme in vivo by using a hairpin autocatalyuc cassette (58). The cis-cleaving autocatalyt~c ca~sette generated a short defined 3' terminus on the HI Y-I speci fie ribozyme and produced a fully catalytically functional ribozyme m VIVO with excellent anti HIY-1 acti\ ity.

4.3 Use of hammerhead and hairpin ribozymes to target

specific RNA transcripts 4.3.1 Design of the ribozyme Certain rules need to be followed in order to design ribozymes for gene-spe'"itic targeting. It is a simple matter fim to identify an appropriate substrate and then to design either a hammerhead or hairpin ribozyme which base pain to the 'arms' of the substrate according to the suuctures given in Figure 7. The hammerhead has two ribozyme-substrate 'arms', helices I and [JJ, wh1ch can be of approximately the same length. The hairpin ribozyme has two ribozyme-substrate helices with very different properties. Helix 2 is fixed at 4 bp while helix I is variable with optimal cleavage rates occurring with length' of 232

David A. Shub. Craig L. Peebles. and Arnold Hampel 6-12 bp depending on the specific nucleotide sequence. Thus for both systems various lengths of these helices must be tested for optimal catalytic efficiency \\ith any particular RNA substrate. It is important to select target sequences ,., hich give reasonable turnover (kc31) \ alues to obviat1! the danger of generating a ribozyme with very IO\\ rates of reaction \\ hkh would give, for all practical purposes, a non-catalytic antisense reaction. For example, the indiscriminate use of unnecessarily long helices between the ribozyme and substrate would lead to such IO\\ off-rates for the product that the rates of reaction would be negligible for any type of gene targeting effect. ln the basic ribozyme-catalysed reaction, both the ribozyme sequence and the RNA substrate are prepared by in vitro transcription. Using a DNA synthesizer and the procedure recommended by the manufacturer, synthesize DNA templates corresponding to the complements of the desired ribozyme and of the RNA substrate. Also include the 17 nt promoter of T7 bacteriophage (sequence 3 '-ATTATGCTGAGTGATAT-5 ') up tream of the sequence to be transcribed. Although transcription will start immecliatel}' folio'' ing nucleotide 17, se.,eral additional nucleotides downstream are also part of the promoter. It is suggested that nucleotides 18, 19 and 20 be chosen as CCC for the ribozyme template and CGC for the substrate template. This start the ribozyme transcript with a 5 'GGG and the substrate with 5' GCG. When in vitro transcription is carried out using [ a-32P] CTP substrate (63), this will ensure at least one labelled nucleotide in the substrate for detection purposes. FoUowing in vitro transcription of these DNA templates using T7 RNA polymerase (see Protocol2 and also Volume I, Chapter I, Protocol4 and Chapter 2. this volume, Protocol 4) purify the 32P-Iabelled substrate and ribozyme on 200Jo polyacrylamide/7 M urea gels (9) using a ppropriate size marker standards. Locate the appropriate radioactive RNA transcripts by autoradiography and elute the RNA (Volume I, Chapter I, ProtocolS, Volume I, Chapter 4, Prowcol II). To ensure the identity of the RNA, it can be sequenced directly with RNA base-specific enzymes. Finally carry out the ribozyme-catalysed cleavage reacuon and analyse the products by denaturing polyacrylamide gel electrophoresis. Details of tbis sequence of investigation are given in Protoco/4.

''ill

Protocol 4 . Carrying out a ribozyme catalytic reaction with

hammerhead or hairpin ribozymes Equipment and reagents • DNA synthes1zer and appropnate nucleotide monomers for the template sequences to be synthesized (ribozyme and RNA substrate), see the manufacturer's recommendations

• T7 RNA polymerase and other reagents for in vitro transcnption as described in Protoco/2 or as in Volume I, Chapter 1, Protocol 4 and Chapter 2, this volume , Protocol 4

• (ot-32P]CTP (specifiC act1v1ty greater than 3000 C1/mmoll

Continued 233

7: Ribozymes Protocol 4 .

Continued

• 20% polyacrylamide 7.0 M urea gel and other materials for denatunng electrophoresos as descnbed on Volume I. Chapter 1. Proroco/ 8 and Volume I, Chapter 4, Prorocol 11

• 0 .5 ml m1crocentrifuge tubes and pestles desogned to l •t IntO microcentrifuge tubes

• Extractoon buffer 10.5 M arnmomum acetate pH 7 0, 2 mM Na EDTA 0 5mg ml SDS J

• 4 x cleavage buffer (0 16 M Tris-HCI , pH 7 5. 48 mM MgCI2 , 8 mM sperm1dmel

• 5 mg 1ml tRNA (yeast)

• Speedvac concentrator

• 70'-b ethanol, 2 mM Na-EDTA • TE buffer 110 mM Tns-HCI . pH 8.0, 1 mM EDTA l

Method 1. Synthes1ze the appropnate T7 phage promoter-ribozyme and T7 phage promoter-substrate templates using the DNA synthesizer and the manufacturer's protocol. 2. Separately transcribe the templates using punfied T7 RNA poly merase and [ a-32P) CTP follow1ng the methods described m Protocol 2 or as in Volume I, Chapter 1, Protocol 4 or Chapter 2, this volume, Prorocol4. 3. To purify the transcripts, run the products of each transcnption react1on on a 20% polyacrylamide/7 M urea gel beside appropriate radioactive stze markers as described in Volume I, Chapter 1, ProtocolS, or Volume I, Chapter 4, Protocol 11. 4 . Autoradiograph the g1el for less than 1 min to detect the 32P labelled transcripts (e.g. using Kodak X-Omat film) 5 . Excise the appropriate· bands from the gel and place each gel fragment in a 0.5 ml microcentrifuge tube . 6 . Add 0 .35 ml of extraction buffer to each tube and crush the gel fragment ustng a pestle. 7 . Shake the samples 1or 20 m1n and then centnfuge the tubes tn a microcentrifuge for 5 min. 8 . Carefully remove the supernatants into 1. 5 ml microcentnfuge tubes and add 1 111 of 5 mgl ml tRNA and 2.5 vol. ethanol. Mix well and leave the tubes at - 20 °C overnight or at - 70 °C for 20 min to prectpitate the RNA. 9 . Recover each RNA by centrifugation at 10000g at 4 °C.

tn

a microcentrifuge for 30 mtn

10. Wash each RNA pellet to remove traces of SDS, which will otherwise inhibit the ribozyme r eaction, by simply rinsing the surface of the pellet and the inside of th1~ tube with 70% ethanol. 2 mM EDTA and briefly centrifuging as in step 9 . 1 1. Repeat step 10 and dry the RNA pellets in a Speedvac concentrator 12. Quantify the yields by Cerenkov counting as described in Volume I, Chapter 1, Section 2 . 3.4 . Adjust the final concentrations to 40 fmol 11JI (ribozymel and 400 fmoi/IJI (RNA substrate) by dissolving the RNA pellets in the appropriate volumes of TE buffer.

Continued 234

Dm·id A. Shub, Craig L. Peebles, and Arnold Hampel Protocol 4 . Continued 13. For the ribozyme cleavage reaction, add the followmg reagents, in order to a 0.5 ml microcentrifuge tube on ice and mix. • H2 0 • ribozyme

2tJI 2 1-11

• RNA substrate 2 1-11 14. For the zero t1me control, remove 31JI into a separate tube containing 3 IJI of formamide-dyes mixture. 15. Heat the rema1n1ng 3 IJI of ribozyme reaction mixture to 80 ° C and then cool it to 25 °C. Add 1 ~JI of 4 x cle!avage buffer and mix. Incubate the sample at either 37 °C or 25 °C for a time penod which w1ll give significan t cleavage, i.e. at least 10%. Normally this will be 10-30 min. The choice of incubation t emperature is dependent on the intended use of the ribozyme. If 1t is a mechanistic study or if the Intended use is for gene targeting 1n plants, 25 °C 1s used. If the ribozyme is to be developed for mammalian gene targeting experiments, then 37 °C is used. 16. Add 41JI of formam1de-dyes mixture to the ribozyme reaction (step 1 51 and separate the products of the ribozyme reaction and the zero time control (step 141 on a 20% polyacrylamide/7 M urea gel (step 31. Auto· rad1ograph the gel and cut out the appropnate bands (steps 4 and 51. 17. Ouant1fy the RNA products by Cerenkov counting (step 1 2) and determine the percentage cleavage of the substrate, after taking account of the zero time control value (step 141.

4.3.2 Determination of the kinetic prurameters for the ribozyme reaction Once the reactivity of the basic ribozyme/ substrate reaction has been established, more detailed kinetic analyses can be done. The Michaelis constant (Km) and the turnover number (kca1 ) are determine·d by carrying out the basic ribozyme reaction (see Protoco/4) over a range of substrate concentratiom both abo'e and below the Km and for times suitable to give initial velocities (64). At least two time points are needed, and no more than 1007o of the substrate should be ciea\ed to ensure that initial rates can be calculated. These mitial \elocities (v;) are then plotted as a function of v/ [substrate]. The Km is calculated from the negati\e slope, and kcaJ is calculated by dividing the y mtercept by the ribozyme concentration. Suitable ribozymes for gene targeting should have catalytic efficiencie (k, 31 Km) in the range of 5-30 mm 1 1-1M- 1• 4.3.3 Use of selected suitable riboz.ymes for gene targeting Upon completion of the studies to identify a useful ribozyme, that is, one which has a suitable catalytic efficiency, the ribozyme can then be used for gene targeting. The specific method of delivery and expression of the ribozyme in vivo will be dependent on the gene being uargeted and the system being studied. For certain applications, the use of awtocatalytic cassettes to terminate the ribozymes 111 vn•o following expression are useful (Section 4.4). 235

7: Riboz_rmes

S' Cassette

5' --c.=:=::::::J3·

l...._...) short ribozyme-derived sequence

hammerhead

3' Cassette 5

(b)

desired transcript

5' c===:::r--3'

l...._...)

3

short ribozyme-derived sequence hammerhead

5'

(c)

desired transcript

5' c===:::r--3

y

short ribozyme-derived sequence hairpin Figure 8 . Transcrcptional term•nat•on cassettes using the hammerhead and hairpin nbozvmes Ia) hammerhead rcbozyme to give 5' termination, lb) hammerhead ribozyme to glve 3 ' termmation, and lcl ha•rprn robozyme to gove 3' termmatoon. In each case, the arrow marks the site of autocatalvttc cleavage

4 .4 Use of autocatalytic cassettes to generate transcripts with defined 5' or 3 ' termini Hairpin and hammerhead ribozymes may be used for the generation of defined 5' or 3' termini on RNA transcripts through the use of cis-clea\ ing autocatalytic cassettes (59-62). These autocatal>1ic cassette~ are created by simply forming loops between the substrate and ribozyme such that they are on the same transcript as illustrated in Figure 8. Upon completion of the in vitro 236

DO\•id A. Shub, Craig L. Peebles. and Arnold Hampe/ transcription of the cassette (see Proroco/4). it \\ill auwcatalytically clea\e to generate a defined terminus. It is imponant to note that the termini generated are a 5 '-hydroxyl group and a 3' terminus with a 2', 3 cyclic phosphate group To use an autocatalytic cassette, it is cloned either in front of the desired transcript (to give a defined 5' terminus) or following the desired transcript (to give a defined 3 · terminus) as sbO\\ n in Figure 8. Among direct applications for cis-acting autocatal} tic cassettes is the generation of defined 5 and 3 ' termini on ribozymes designed to be delivered for gene targeung (58, 62). That is, the ribozyme to be delivered by in \ illo transcription would have termination casseues at either or both of the 5' and 3' termini. A cassette on the 3' terminus of the ribozyme is especially useful, because then the newly-transcribed ribozyme can be processed with the elimination of the requisite 3' poly(A) tail and normal transcriptional termination sequences (58). The resulting ribozyme is then very small and perhaps more likely to behave as an optimally functioning catalyst in \•ivo.

Acknowledgements The work m Section 2 was supported by grams from the IH to D. A. S. The work in Section 3 was supponed by grants from the American Cancer Society and NSF to C. L. P. , who thanks them for their suppon. C. L. P . also thanks P. S. Perlman for scientific collaboration throughout this project, J. S. Franzen for the original drawings adapted into several of the figures, and M. Zhang for scientific and technical assistance. The work in Section 4 was supported by grants from the Biotechnology Research and Development Corporation and NIH to A. H. A. H . thanks M. Altschuler and R. Tritz for technical and sciemi fie assistance.

References I. Cech, T., Zaug, A., and Grabo\\Ski, P. (1981). Cell, 21, 486 . 2. Guerrier-Takada, C .. Gardiner, K., \Iarsh, T., Pace, 'J, and Altman, S. (1983). Cell, 35, 849. 3. Kruger, K.. Grabowski, P. J., Zaug, A. J., Sand~. J., Gomchling, D. E., and Cech, T R. (1982). Cell, 31, 147 4. Michel. F .. Jacquter, A., and Dujon, B. (1982). Biochim1e, 64, 867 5. Michel, F. and Westhof. E. (1990). J ..\fol. Bioi., 216, 585. 6. Reinhold-Hurek, B. and Shub, D. A. (1992). Nawre, 357, 173. 7. Lambowitz, A.M. and Perlman. P. S. (1990). TIBS, 15, 440. 8. Garriga, G and Lambowitz, A.M. (1984). Cell, 39. 631 9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular donmg. A laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor i'. 't. 10. GotL, J. M., Shub, D. A .. and Belfort, M. (1986). Cell, 47, 81. 11. Birnboim. H. C. and Doly, J. (1979). Nucleic Acids Res.• 7. 1513. 12. Cech, T. R. (1990) Amw. Rev. Biochem., 59. 543.

237

7 Ribozymes 13. \IIebel, F .. Umesono. K .. and Ozeb.i, H. (1989). Gene, 82, 5. 14. Cech, T. R. (1988). Gene, 73, 259. 15. Burke, J. 'vi .. Belfort, M., Cech, T. R., Davies, R. W., Schweyen, R. J., Shub, D. A .. Szostab., J. \\ .. and Tabab., H. F. (1987} \ucle1c Acids Res.. 15, 7217. 16. Peebles, C.L., Perlman, P. S., 'vlecklenburg, K. L., Petnllo. M. l., Tabor. J. H .. Jarrell. K. A., and Cheng, H . L. (1986). Cell, 44, 213. 17. van der Veen. R., Arnberg, A. C., Vander Horst, G., Bonen, L., Tabab., H . r. and Grhell. L. A (1986) Cell, 44, 225. 18. Schmelzer, C. and Schweyen, R. J. (1986} Cell, 46. 557. 19. Kuck, U., Godehardt, 1., and Schmidt, U. (1990). Nucleic Ands Res., 18, 2691 20. Schmidt, U., Riederer, B., Marl, M., Schmelter, C., and Stahl, U. (1990). EMBOJ. , 9, 2289 21. Hebbar, S. K., Belcher, S. M ., and Perlman, P. S. (1992). Nucleic Actds Res., 20, 1747. 22. Jacquier, A. and Michel, F. (1987). Cell, 50, 17 . 23. Jacquier, A. and \lichel, F. (1990}. J. Mol. Btol.. 213, 437. 24 Michel, F. and Jacquier, A. (1987) Cold Sprmg Harb. Symp. Quant Bioi. 52, 201. 25. Jarrell, K. A ., Dietrich, R. A., and Perlman , P. S. (1988). Mol. Cell. Btol. 8 , 2361. 26. Schmelzer, C. and Muller, M. W . (1987). Cell. 51, 753. 27. van der Veen, R., Kwakman, J. H., and Grivell. L.A. (1987). EMBO J., 6. 38~7. 28. Jacquier , A. and Jacquesson-Breuleux, N. ( 1991). J. Mol. Bioi., 219, 415. 29. Peebles, C. L., Benatan, E. J., Jarrell. K. A. and Perlman, P. S. (1987). Cold Sprmr: Harb. Symp. Quam. Bioi., 52, 223. 30. Koch. J. L, Boulanger. S.C., Dib-HaJj, S. D., Hebbar, S. K., and Perlman. P. S . ( 1992). Mol. Cell. Bioi., 12, 1950. 31. Wallasch, C., Marl, M .• Niemer,l., and Schmelzer, C. (1991). Nucleic Acids Res. 19, 3307. 32. Jarrell, K. A. (1987). Ph. D. Di\sertatlon, Ohio State Univer~ity, Columbus, Ohio. 33. rranzen, J. S., Zhang, M., and Peebles, C. L. (1993). Nucleic Acids Res.. 21, 627. 34. Jarrell. K. A., Dietrich, R. C., and Perlman, P. S. (1988). Mol. Cell. Bioi., 8, 2361 . 35. '\.1uller, \1 . \\ , Schweyen, R. J., and Schmelzer, C. (1988). Vue/etc Ands Re\., 16, -383. 36. Jacquier, A. and Rosbash, 1\1. (1986). Sctence, 234, 1099. 37. Choquet, Y., Gold\chmidt-Clennom, M., Girard-Bcou, J., Kuck, U., Bennoun, P ., and Rochaix, J. D (1988). Cell. 52. 903. 38. Hildebrand, M .. H allicb., R. P .. Passavant, C \\ . and Bourque, D P (1988}. Proc. Nat/ Acud. Sci. USA, 895, 372. 39. Kohchi, T., Ogura, Y., Ume~ono, K.. Yamada, Y., Kamano, T., Ozeki. H .. and Ohyama. K. (1988). Curr Genet., 14, 147. 40 Gold~chmidt-Clermont, M., Choquet, Y. Girard-Ba.cou. J., \lichel, F Schirmer Rah1re. ~!.,and Rocha1x, J . D (1991). Cell, 65, 135. 41. Augu~tm, S., Muller, \11. W., and Schweyen, R J. (1990). !Vature, 343, 383 . 42. Morl. M. and Schmelzer. C. (1990). Nucleic Actds Res.. 18, 6545 43. Forster, A. and Symons, R. (1987) Cell. 49, 211. 44. Uhlenbeck, 0. ( 1987). \'awre, 328, 596. 45. Hampel, A. and Tritz, R. ( 1989). Biochemistry, 28, 4929. 238

Da\·id A Shub. Craig L. Peebles. and 1\rnold Hampel ~6.

47. JS. 49 50.

51. 52.

53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63 ~-

Hampel. A., Tritz, R., Hie~\. \1 .. and CrU7. P. (1990) Nucleic Acids Re~. 18. 299. Haseloff. J. and Gerlac.:h. \"- ( 1989). Gene, 82, 43. Buz.a~an, J • Gerlach. \\ ., and Bruenmg. G. (1986). Vawre. 323. 349 h.eese, P. and Symons, R. (1987) In Virotds und l'iroid·like pathogens (ed. J. S. Semanc1k), p. I. CRC Press, Boca Raton, fL. Bruemng. G., Buzayan. J .• Hampel, A., and Gerlach,\\. (1988). In R'\'A genetics; ~ olume II· re1rowruses, l"iroids, and RAA recombination (ed. E. Domingo, J. Holland. and P L. Ahlqui\1), p. 127. CRC Press, Boca Raton, FL. Ros~i. J and Sarver, N. (1990). TIBTECH, 8, 179. Sarver, N .. Johnston, M., Hampel, A., Zaia, J , Cantin, E .. Chang, P • and Rossi. J. ( 1990). In Gene re~ula11on and A IDS: transcnpflonal acfil·utton, retro1·intSes and pathogenesis (ed. Tak1s Papa~). Gulf Publishing Co., Houston, T'\. p. 305. Fedor, \1. and Uhlenbeck, 0. (1990). Proc. Nat/ Acad. Sci. USA. 87, 1668 Pieken, W., Olsen, D., Benseler. F., Aurup, H., and Eckstein, r. (1991). Science, 253, 314 Sar\er, "'·· Cantm, N., Pairoj, C., Zaia, J., Ladne, P .. Stephens, D., and Ros~i, J. (1990). Sctence, 247, 1222. Haseloff, J. and Gerlach, W. (1988) Nature, 334. 584. Scanlon, K., Jiao, L., Funaro. T., Wang. \V .. Tone, T , Rossi, J., and h.ashaniSabet, M. (1991). Proc. 1\ut/ Acad. Set USA, 88. 10591. 0Jwang, J., Hampel, A., Loone), D, \\-ong-Staal, F., and Rappaport, J. (1992). Proc. Nat/ A cad. Sci. USA, 89, 10 802. Dz•anott, A. and Bujars~i. J. (1989). Proc. !Vat/ Acad. Sci. USA, 86, 4827 Grosshans. C. and Cech. T. (1991). \ucleic Actds Res., 19, 3875. Taira, K. , \1ansanao, 0 .. Shm\hl, H. , Maeda, H., and Furu~awa, K. (1990). Protem Engineering, 3, 733. Alt~chuler, M., Tritz. R .. and Hampel, A . (1992). Gene, 122, 85. Milligan, J., Groebe. D., \\'itherall. G., and Uhlenbed... 0. (198-) Vucleic Acids Res.• 15, 8783. Fersht, A. (1977). En;;yme srrucwre and mechamsm. W. H. Freeman and Co .. San Francisco. CA.

239

l

Suppliers of specialist items

American Type Culture Collection (A TCC), I 230 I Park La\\0 Drive, Rockville, MD 20852, USA. Amersham International PLC, Lincoln Place, Green End, Aylesbur}, Bucks HP20 2TP, UK. Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, lL 60005, USA. Amicon Ltd, Upper Mill, Stonehouse, Gloucester GLIO 2BJ, UK. Amicon Division, WR Grace & Co., 72 Cherryhill Drive, Beverley, MA 01915-1065, USA. Applied Bios) tern Inc., 850 Lincoln Center Drive, Foster City, CA 94404, USA. Applied Biosystems Ltd., Kelvin Close, Birchwood Science Park North, Warrington WA3 7PB, UK. J T Baker Chemicals Inc., 222 Red School Lane, PO Box 492, Phillipsburg, NJ 08865-9944, USA. J T Baker UK Ltd., Wyvols Court, Basingstoke Road, Swallowfield, Nr Reading, Berks RG7 I PY, UK. Barnstead Instrument Co. , 2555 Kerper Boulevard, Dubuque, lA 52001, USA. Beckman In truments UK Ltd., Progress Road, Sands Industrial Estate, High Wycombe, Bucks. HPI2 4JL, UK. Beckman Instruments Inc .. PO Box 3100, 2500 Harbor Boule\ard, Fullerton, CA 92634, USA. Becton Dickinson Lab~ are, 2 Bridgewater Lane, Lincoln Park, NJ 07035, USA. Becton Dickinson Ltd. , Bet\\een TO\\ OS Road, CO\\ le}, Oxford OX4 3LY. UK. BeUco Glass Co., PO Box 8, 340 Endrido Road, Vineland, NJ 08360, LSA. c/ o Scientific Laboratories Supplies, Unit 27, Nottingham South and Wilford Industrial Estate, Ruddington Lane, Wilford, Nottingham NG II 7EP, UK. Bethesda Research Laboratories; see Gibco BRL. Bio-Rad Laboratories Ltd., Maylands Avenue, Hemet Hempstead, Hens HP2 7TD, UK.

Appendix 1

Bio-rad Laboratories, Di\·ision Headquarters, 3300 Regatta Boule\·ard. Richmond, CA 94804. USA. Bodman, Aston, PA 19014, USA. Boehringer Mannheim GmbH Biochemica, PO Box 31 01 20, D-6800 ~1annheim, Germany. Boehringer Mannheim UK (Diagnostics/Biochemical!>) Lid., Bell Lane. Lewes, East Sussex BN7 lLG. UK. Boehringer Maonheim Corporation, BiochemtcaJ Products, PO Box 5041-t, lndianapohs, IN -t6250-0413. uSA Braun (B. Braun l\1elsungen AG) , PO Box 120, D-3508 \.telsungen, German) Braun (B. Braun Medical Ltd) , 13- 14 Farmborough Close, Aylesbur} \'aJc Industrial Park, Buck~ HP20 JDQ, UK. Brinkman Instrument Co., Cantigue Road, Westbury, "'Y 11590, USA. c/ o Chemlab Instruments Ltd., Hornminster House, 129 Upmin~ter Road, Hornchurch, Essex, UK. Calbiochem, PO Box 12087, San Diego, CA 92112-4180, USA. Calbiochem-Novabiochem (UK) Ltd., 3 Heathcoat Building, Highfields Science Park. University Boule\ard, Nottingham NG7 2QJ, UK. Cornjng Medical and Scientific Glass Co .. Medfield, \1A 02052, USA. Ciba Corning Diagnostics, Colchester Road, Halstead, Essex C09 2DX. UK. Difco Laboratories Ltd., Central Avenue, East Mole ey, Surrey KT8 OSE, Ll . Difco Laboratories, PO Box 331058, Detroit, Michigan 48232-7058, USA Du Pont Co. (Biotechnolog) Systems Di\·ision), PO Box 80024, Wilmington, DE 19880-0024, USA. Ou Pont UK Ltd., Wedgwood Way, Stevenage, Hens SGI 4QN, UK. Eastman Kodak Co. , PO Box 92822, LRPD-1001 Lee Road, Rochester, NY 14692-7073, USA. c/ o Pha~e Separations Sales, Deeside Industrial Park, Deestde, Clw)·d CH5 2NU, UK. Falcon; contact Becton Dickinson. Fisher cientific Co .. 50 Fadem Road, Springfield, NJ 07081, USA. Flo\\ (ICN Flow), Eagle House, Peregrine Business Park, Gomm Road, High Wycombe HP13 7DL, UK. Flo\\ (lC:-.. Biomedjcallnc.), 3300 Highland Avenue, Costa \lesa. CA 92626, USA. Fotodyne Inc. , 16700 West Victor Road, New Berlin, Wisconsin. USA. Gibco BRL (Life Technologies Ltd), Trident H ouse, Renfrew Road, PaiskY PA3 4EF, UK. Gibco BRL (Life Technologies Inc.), 3175 Staler Road, Grand Island, :-. Y 14072-0068, USA. Gilson France SA, BP 45, 72 rue Gambeua, 95400 Vilbers le Bel France. c o Anderman L!d .. 20 Charles Street, Luton, Beds LU2 OEB. L"K. Hoefer Scientific Instruments, PO Box 77387-0387, 654 Minnesota Street. San Francisco, CA 94107, USA. Hoefer UK Ltd., Newcastle, Staffs ST5 OTW, UK. 242

Appendix 1 H~Cione Laboratories, 1725 State Highway 89-91, Logan, UT 84321. USA. IBI (International Biotechnologies Inc.), PO Box 9558, e\\ Haven CT 06535, USA . IBI Ltd., 36 Clifton Road, Cambridge CBI 4ZR, UK. ICN Biomedicals; see Flow (ICN). ISCO Inc., PO Box 5347, Lincoln, NE 68505, USA. c/ o Jones Chromatography Ltd., Tir-y-Berth Industrial Estate, Ne\\ Road, Hengoed, !\tid Glamorgan CF8 8AU, UK. lsolab Inc., PO Box 4350, Akron, OH 44398-6003, USA. c/ o Genetic Research Instruments Ltd., Gene House, Dunmo\\ Road, Felstead, CM6 3LD, UK. Kimble Products, 1022 Spruce Street, Vineland, NJ 08360, USA. Kodak; see Eastman Kodak. Kontes Glass Co., Vineland, NJ 08360, USA. c/o Burkard Scientific Sales, PO Box 55, Uxbridge, Middx UB8 2RT, UK. M.A. Bioproducts (Microbiological Associates), Biggs Ford Road, Building 100, Walkersville, MD 21793, USA. c/ o Lab lmpex Ltd., Waldergrove Road, Teddington, Middx., T\Vll 8LL, UK. Macberey Nagel, Postfach 307, Neumann-Neanderstrasse, D-5160 Duren, Germany. c/ o Camlab Ltd., Nuffield Road, Cambridge CB4 ITN, UK. Millipore lntertecb, PO Box 255, Bedford, f..IA 01730, USA. MiJJipore UK Ltd., The Boulevard, Blact...moor Lane, Watford, Hens WDI 8YW, UK . Nalge Co., PO Box 20365, Rochester, NY 14602-0365, USA. c/o FSA Laboratory Supplies, Bishop Meadow Road, Loughborough, Leics. LEI I ORG, UK. New England Biolabs (NBL), 32 Tozer Road, Beverley, fvlA 01915-5510, USA. New England Nuclear (NE~), Du Pont Co., NEN Research Products, 549 Albany Street, Boston, r-.1A 02118, USA. DuPont UK Ltd., Wedgwood Way, Stevenage, Herts SGl 4QN, UK. Pharmacia Biosystems Ltd. (Biotechnology Division), Davy Avenue, Knowlhill, Milton Keynes, MK5 8PH, UK. Pharmacia-LKB Biotechnology Inc. , PO Box 1327, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA. Pierce, PO Box 117,3747 North Meridan Road, Rockford, 1L 61105, USA. c/o Life Science Labs Ltd., Sedgewick Road, Luton, Beds LU4 9DT, UK. Pierce Europe BV, PO Box 1512,3260 BA Oud-Beijer1and, The Netherlands. Promega Ltd., Delta House, Enterprise Road, Chilworth Research Centre. Southampton SOl 7NS, UK. Promega, 2800 Woods Hollo'' Road, ~ladison, WI 53711-5399. USA. Qaigeo Inc., Studio City, CA 91604, USA. c/ o Hybaid Ltd., 111-113 Waldegrave Road, Teddington, Middx T\\ II 8LL. UK.

243

Appendix 1 Sartorius AG, Postfach 32-43, Weender Landstrassc 94-108, D-3400 Gottmgen, Germany. artorius Ltd., Longmead, Blenheim Road, Epsom, Surrey KT9 9Q , UK. Sartorius ~orlh America Inc. , 140 Wilbur Place, Bohemia, Long Island, N"\ 11716, USA. Schleicher & Schuell, Postfach 4, D-3354 Dassell, Germany. c/ o Anderman & Co. Ltd. , 145 London Road, Kingston-upon-Thames, Surrey KT2 6NH, UK. Searle; contact Amersham. Sigma Chemical Co. Ltd., Fancy Road, Poole, Dorset, BH17 7NH, UK. Sigma Inc., PO Box 14508, St Louis, MO 63178, USA. Sorvall; contact Du Pont. Squibb Pharmaceuticals , I Squibb Drive, Cranberry, NJ 08Sl2-9S79, USA. Stratagene Inc., 11011 North Torrey Pines Road, La Jolla, CA 92037, USA. Stratagene Ltd., Unit 140, Cambridge Innovation Centre, Milton Road, Cambridge CB4 4FG, UK. United tales Biochemical (USB) Corporation, Box 22400, Cleveland, OH 44122, USA. c/ o Cambridge Bioscience Ltd., 25 Signet Court, Stourbridge Common Business Centre, Swans Road, Cambridge CBS 8LA, UK. Unhersil)' of Wisconsin, Genetics Computer Group, University Avenue, Madison, WI 53706, USA. UV Products lnc., S100 Walnut Grove, San Gabriel, CA 91778, USA. UV Products Ltd., Science Park, Milton Road, Cambridge CB4 4BN, UK . Van Waters & Rogers, PO Box 6016, Cerritos, CA 90702, USA. Virtis Co. Inc., Route 208, Gardiner, NY 12S2S, USA. c/ o Damon/ 1EC (UK) Ltd., Unit 7, Lawrence Way, Brewers Hill Road, Dunstable, Beds. LU6 lBD, UK. VWR Scientific Product , PO Box 7900, San Francisco, CA 94120, USA. Wako Pure Chemicals, Dosho-Machi, Osaka, Japan. Waring Commercial, c/ o Christison Scientific Equipment Ltd., Albany Road, East Gateshead Industrial Estate, Gateshead NE8 3AT, UK. Whatman cientific Ltd., Whatman House, St Leonards Road, Maidstone, Kent ME16 OLS, UK. Worthington Biochemical Corporation, Halls Mill Road, Freehold, NJ 01728. USA. c/ o Cambridge Bioscience Ltd., 25 Signet Court, Stourbridge Common Business Centre, Swans Road, Cambridge CBS 8LA, UK.

244

Contents of Volume I 1. Synthesis and purification of RNA substrates Benoit Chabot

2. Characterization of RNA Paula J. Grabowski

3. Splicing of rnRNA precursors in mammalian

cells Jan C. Eperon and Adrian R. Krainer

4. Isolation and characterization of ribonucleo-

protein complexes Angus I. Lamond and Bnan S. Sproat

5. Analysis of ribonucleoprotein interactions Cindy L. Will, Berthold Kaster, and Reinhard Luhrmann

6. Analysis of pre-mRNA splicing in yeast Andrew Newman

Index \!any of thl' nandard me/hods for p"parallon OJ R.\ ..J Jubclle\, riboty me for generating 5' and 3 R'A termini ~36-i

bacteriophage R'A polymera>C>, ior in ntro transcription of capped mRI\A 43-4 guide RNA' 91 pre-mR' 1\ for J end-pro.:e"ing 1-3 pre-rR:-..\ 1~-il pre-tR!"A 11!1-5 'ubstrate\ for cr;.ptic R'-"<~'e 100-1 51'1' also 111 I'IITO tramcription ba'e modification~ in tRNA 199-:!06 calf thymu,, 3' cnd-proce~'ing factor' :!9-33 cappang enLyme,, <ee guanylyl transfcra\e, R:>.A triphosphata~e capping ol .:ellular mR~A by enzymic recappin~ -li"-8 by m l'ttro tran,aiption -13--1 \iral R:-: \ U\ing imect cytopla,mic polyhedrosi\ 'ITUS 39-41 using reo' irus 41-2 using \accinia \irus -1:!-3 cap structures in mRSA chemical di' er,il\ 35-6 enzymic clea\ag~ 55-6 formation 111 \'11'0 36-7 radiolabelling by decapping and recapping -15-8 by "' I'UTO transcription -13-4 by 0-methylation 51-1 0\enie\\ 38-9, -15 b\ periodate oxidation and boroh}dride reduction 39--13 by \iral transcription 39-43 role in mRNA ~tabilization 3 separation of. by ion c.\change chromatograph) 57-9 paper chromatography 59-61 paper electrophoresi' 62-3

TLC on PEI-cellulo~c 61-2 two-dimcmional chromatography 64-5 cell,, t1"ue culture. extract prcparauon. for mRI\A turno,er 11-1-19 . .\1!1! also Hela cell cis-,plicing 225-6 clea,age and polyaden)lation ol mR'\A m l'tiTO, coupled 21-3 cleavage and polyadenylation \pecificity factor (CPS!-) as~ay 32-J interaction "ith A .·\ UAAA 'equence 6--;. 15 in' oh·ement in 3 ' cnd-proce,~ing of mR:-.:A 1-:!, 24 purification from calf th) mu' :!9-30 Hela cell~ .?~ -R 'eparation from poly(t\) polymera~c 21-9 clea,age or mR'\A 1-2. 21-" rRNA I J5· 8, 149-51, 153-5 CPSf, see clca,age and polyadcnylation 'pccilidty fac10r CrO\~-Iinkin~

R!'.A-prouin 160--1 RNA-R:-.A 142-J Iee also Volume I crypuc RNa\e 96. 100-1 cy1oplasmic e'\tract\ for mR'\ .\ turnover III-I-I. 119-26 for rR:-.A proce~smg 150-5

decapping or RNA chemical 45-7 enzymic 52-5 deletion analy'i' of rR:"/A pro.:e"ing 155 DHFR-Iigase fu,ion protdn, ewr~sion 191-4

edning, RNA. in kinetoplast mitochondna as~a) of enqmes involved cry·ptic RNase I 00-1 gR'\;A:mRr-;A chimaera-forming actiHt} 101-3 O\ en ie" 96-7

Index e\amrle' 24-5 e\tract preparation 4-6 general consideratiom ~-J interpretation of data :?4-~ non-,pec.:ific polyaden)lation !4 o'er~ ie" :? l. :?6 polyadenylation comple' formation anaJy,ts by electrophore~i\ "-10 analy~t~ by modification-interference

eduing. R:-.A, m l.inetopla't miux:hondria (COni.)

ao,say of enzyme~ in,ohed (mnr. ) R'A liga~e 99-100 term mal uridyl) I tramfera~e 97-9 choice of experimental organi<m 71-.:: clea\age-ligation model 69-70 doning of L. torenrolue 7~ enzyme cascade model 69-70 gRNAs hybnd selecllon 88- 90 identification 94-6 in gRNA:mRNA ch1macras 69-70,

90, 96

in l'ltro transcription 91 gro" th and maintenance of L. torentolae kinetoplast DNA (1.0:".-\) i>olation 76-8 ,chizodeme typing of proto7oa 78-9 sl!e also kinetoplast D~A ~inetopla;,t mitochondrial lra"ion 69-11. 81-5, see also mitochondria kinetoplast RNA (kRNA) ·~lation 87-8 "onhern blot anal}\is 88 occurrence 69-71 PCR amplification of partially edited mRNAs and gRNA"mRNA chimaeras

90, 92-3 trum-c;terification model 69-70, 90, 96, 101-2 tvpes of editing 69-71 electron microscopy of rRNA synthesis 143-4 electrophoreSIS, see agarose (name) gel clectrophore.>l\, formaldehyde-agarose gd electrophoresi,, paper electrophoresis. polyacrylamide urea (denaturing) gel electrophorc
15-21 analy~is

by RNa~e protection 10-14 background 6-7 basic procedure 7-10 Lime course 9-10 polyadenylation of RNA 23-4 RNA substrates 2-~. 15-16 3 end-processing of mRf\A m l'ivo 1-2. 33 3 end-processing of r!U'.A 135-7, 169--o 3 end-processing of tRNA 175, 183, 188-9 5 end-proce•"ing of mRNA, .~ee capping, cap '>tructure> 5' end-proc~sing of rR"-A 135-;, 148-51.

153-5

5'

end-pr~ingoftRN .-\

175. 183, 186, 188-9

enzyme ca,.:.lde model. R'A editing 69--o non-binding sequenc~ 10 grour II ribozymes 219-20, 225, 228 e'press10n of DHFR-Iiga<e fusion protein 191-~

e\lernal transcribed spacer. pre-rRNA

135-9, 142, 144. 148, 150-1, 155, 157-60 extract preparation. for RNA processing, ~ee lysolecithin e\lracts, nuclear e\tracts, post-nuclear ~upernatant, po>t-polysomal. ~upcrnatam, processing e\lract'; see ulsu Volume I

formaldehyde-agaro'e gel ele.:trophore'l~ 10 'onhern blotting of I.R'
gel-shift analysis of protein-rR~A Interactions 156-' gene targeting with hammerhead and ha1rpm ribozymes D5-6 globin mRNA turnover 121-30 gradient centrifugation, 5ee t~Op)·cnic demit) gradients, rate zonal centrifugauon gRNA:mRNA chimaeras amplification b} PCR 90 analy,is by RT -PCR 103 formation in mitochondnal extracts 96-7.

101-J role m R'
248

Index group I intron ribozyme> detection 213-15 mechan i~m 2 II -13 occurrence :! I I group II intron riboz)mL"S C'ts-~plking 125-6 d1•11nction from group I imron nbol)me~ 217-:!0 idemificat1on of 217-19 occurrence 217- I 9 potential u.1es 219-21 reverse splicing and transpo~iuon reactions 228-30 secondnr~ muctures in 219-20 self-splicing of ba>ic procedure 222-3 mechanism 218-20 modification of reaction condition' 224 tran,cripts for 221-2 spliced e~on reopening reactions 125-7 >pliced junction hydrolysis 225-1 tran,.:npts for, preparation 221-1 tran.r-,phdng reacuons 22~ -S guide RN·\ hybrid 'ele.:t100 Of 88-90 identification b) computer-asmted sequence comparison 94-6 in editing of mitochondrial Rl'.A 69-71, b7 >ynt he>i' b~ m l'itro transcription 91 see also gRNA:mRNA ch1maera1

hammerhead and hairpm ribozymc1 in nutocatal~tic casseues 236-7 design of 232-3 for generating transcripts with defined termini 236-7 in gene targeting 131-6 kinetic' of :!33, 2.35 mechani•m' 130-1 occurrence :no mucture 230-3 Hela cell e:\lracts tor 3 end-processmg 4-6 rR"'A pro.:~sing 150-5 m -G-spec1 f1c pyrophosphatase 53 purification of 3 end-processmg factor' 26-9 heparm-agaro1e chromatography, purification of )east o!ndonuclca1e 189-91 histone mRNA end-proce~1ing 2 turnover 129 hybrid selection of guide RNA' 88-90 rR:-. \ Intermediate' 139-42

imernal transcribed spacer of rR:".-\ I 3b imron-binding ,equence,, m group II ribol)mt~ 219-21 , 215. 228 imrom group I ~elf-~plicing nbol}me> 211-1 7 group 11 self-splicing riboz}mes 217-30 m vitro tran,cription to confirm exi~tence of group I nbozyme~ 215-1? of RNA 'ubstrates capped 43-J for 3' end-processing 2-4 for group II self-~plic ing introm 221-2 for hammerhead and hairpm ribozyme~

233-5 for pre-mRNA turnover 108-9. 121-6, 128-30 pre-rR~A 144-6 pre-tR'\'.\ 181-5 ,-ira! 38-JJ see also Volume I ion nchange chromatograph) of capped oligonucleotides 57- '.1 DHFR-liga'e fusion protein 192-.S poly(A) polymerase and CPSF 27-30 pre-tR"'A >ubstrate 179-80 isopycm.: gradient centrifugation usmg CsCI for kDNA 75-8 for maximcle DNA 7'.1-81 using Renografin or Percoll isolation of l..mctoplast mitochondrial fract1on 82-6 relauve ments 82, '.II

kinetopla't DNA (I..D:-oAI analy~is 78-9. 82 integrit) of 78. 80 holation -6-8 'maxicircle' DNA 15, 79-81 'minicirde· D'\A -s properties 75-6 scbizodc:me t}ping of kinetoplasLid protOLoa 7b-9 transcripts or 85-7 'isualization 78-80 l..metoplasud protozoa cho1ce of specie~ 71-2 editing enzyme,, assa) 96-103 gro"'th and mamtenance n-5 guide RNAs of 8!!-91, 94-6 kDNA preparation 76-81 mitochondrin, isolation 81-5 mitochondrial 1ranscripts 85-8 RNA eduing in 69-71 run-on transcriptiOn 10 mnochondria 111-4

249

Index ~uide R"As lll!-91. 9.1-t'> i\olation 81-.5 ~ DN \ preparauon 15- s I R '- '\ editinf! in 69-'1 run on tramcription .n 91-4 tram.:ript> of b.5-a modification-interference analy,;, of I'Oiy· adenylation comple\e< 15-21, see also Volume I mR:-IA analy-103. <ee also cdilm11 guideR:"-<\ -· end-proce..~ing 1-25, <ee also 3' end·

kme10pla\l RNA !I..R'\AI analy~i' 87-8 compo,ition 85-7. 90 editing 69-; I hybnd ,eJe.:tion of gR'\IA~ from 81!-90 isolation 85-8 PCR amplification of mR'
labelling R:-
proce~smg

io !!R:"'A:mR"'A .:htmacra' 69-iO, 90, 92-3. 96-7. 101-3 polyadenylation 21-33, Jl'l' ul o) endproce,
preparallon II 0- I I ;plicing group 1 introm 211-1 7, 5t'~ ulso group I intron ribo.ryme< group 11 intro"' 217-~0. 'ce also group II intron ribozym~' ree also Volume I stab1l11y regulatOr) factor II ~-.lO ~ubstrate-< for mRNA IUJOO\Cr lOS-II turno\cr in \'tiro of endogenous mR:>:A 108-9. 119-~1. 124, 127-~ extract for, preparation 111-19 of in l'tlro tran~cript' 108-9, 121-6, 12S-30 messenger Rl'
tn \'i\'0

b> ligation 15-16, 99-100 rRNA 139-40 tR A lT-81 by run-on transcription 91-4 see u/so Volume 1 lariat' ele.:trophore;i~ of 221 in group II mtron \elf·;plicing 11~-19 , .!25-6, 128-9 purification 229 ly;oledthin ly
messenger RNase• 112, 119, 1:!2-3 methotrexate-agaro~e chromatography 192-3 methylation of RNA tn vttrn 38-42, 47-8, 51-:! in 1'11'0 35-7 micrococcal nuclease remo,al ol mRNA from poly;omc; 11:!-13 treatmeru of e'tracts 11"-l!l, 12.5-6, 164-5 mitochondria ";th group 1 <elf-splicing intrcon< 211 with group II ;elf-splicing intron~ 217-lb kinetoplast editing enzyme> 96-103 C\tracts for editing !li, 10:!-3

tr~uble\hooting 131-~

see al
!\orthern blot anal}'is of kRNA 88 of snRNAs a ..,ocmted nuh pre-rRr\A

157. 160 see also Volume I

nuclear extracts for 3' end-proces,ing of mR~A o1-6

250

Index rRNA proce~\ing 148-50. 164-9 tR"'A proces~ing 187-9 set> also \ olume I nuclea>e-free carriers for ethanol predpttation 176-7 nuclease PI in analysis and preparation of RI'.A caps 40, 42. 52, 54-5, 62 in identification of tR!'.A splice sites 201-3 nucleolar endonuclease, cleavage of prerRNA 155 nucleolar extract, for rR~A processing 150-5 nucleoude analysiS by TLC on PEl-cellulose 201-4 by two-dimensional chromatography 203, 205-6 nucleotide sequence analysb of gRNAs 94-6 of panially-edited mRNA 90, 92-3

oligonucleottde-directed RNase H digesuoo of RNA in rRNA processing extra.:t~ 165-8 in Xenop11S OOC)tes 168-9 see also Volume I

paper electrophoresis of mRNA caps 62-3 periodate oxidation in decapping mRNA 45- ~ in labelling caps 48-51 poly(A)-binding protein, 10 mRNA turnover 128-30 polyacrylamide (native) gel electrophoreuc analysis of polyadenylation complexes 7-10 polyacrylamide. urea (denatunng) gel electrophoresis assay of gRNA:mRNA chimaeras 102-3 assay of mitochondrial cryptic RNa'e 101 for group II self-splicing introns 221 of pre-tRNA 180-1, 183-5 in scluzodeme analysis of kDNA 78-9, 82 of spliced tRNA 197-9 see also Volume I polyadenylation complexe> analysis by electrophoresis 7 -I 0 modtfication-interfercnce 15-20 RNa\e protection 10-14 formation in extracts 6-10 polyadenylation of mRl'o \ cleavage and polyadenylauon spectficlly factor, purification 26-30, 32-3 in extracts coupled to endonudeolyuc deavage 21-3

interpretation of data 24-5 method and analy\i\ ~~-4 ovenie"' :!1 predea'ed R:\A substrates 23-4 general considerauon~ 2-4 in mammalian celb 1-2 non-speci fie 24 poly(A) polymerase, purification 26-32 ~ubstrate; for 2-4, 15-16 poly(A) polymerase assay 30-2 in 3 end-processing of mR;o.; ·\ 1-2. 24 purification 27-30 separation from CPSF 27-9 polymerase chain reaction (PCR) amplificauon of partially e·dtted mRNA and gRNA:mRNA chimaeras 90,

92-3 see also Volume I polysomes as endogenous substrate feor mRNA turnover 109, 119-21 non-specilic mRNA degradation in 131 preparation II 0-11, 114- 16 riboo,omal salt wash (RS\\) from 116-17 as source of messenger RNase 112 turnover of mRNA in 12'7-8 post-nuclear supernatant mRNA turnover m 123-~· preparation 117-18 treatment with micrococcal nuclea...: 118-19 post-polysomal supernatant (5130) messenger RNases in 119 preparation 114-16 pre-mRNA, pre-rRNA, pre-tRNA, see m \'itro transcription, labelling RNA, RNA ~ubstrates

processing extracts for eduing of mRNA 9~-103 end-processing of mR"lA 4-6, 21-5 mRNA turnover 111-26 rRNA 148-55 tR:-.:A 185-91, 195-9 see also cytoplasmic eMra\'ls, lysolecithin extracts, nuclear extracts. nucleolar e'\tract. post-nuclear •upernatant. post-poly~omal supernatant, and Volume I psoralen, cro~s-hnl..ing of ~n RI'.As and rR:"\-\ 142-3 pyrophmphatase>, in cap analysts 39, 52-6

rate zonal centrifugation, analysis of rRNAprotein associations 15 7-9 reticuloc~te lysate, mRNA mmover in 113, 125-6

251

index re' er-;e 'PI icing of group II intron~ 226-8 ribosomal RN ,-\ pro.:e"ing in ntro anal}m b} cro~\-linling

guanyltransfera>e (capping cnz~ me) 31--8, ~5. 47-b Iigas<' in cdoting of R~A 96, 99-100 in end-labellinl! ot Rl\/\ 15-16 preparation a., fusion protein !Yt-4 in pre-tRNA )plictng 186, 196-7 methyltransferasC) 35-~. ~7- 11. 51-:! pol} merase, .see ba.:terioph311e R:-.' t\ poly mera~e... in ntro tramcnpuon

160-~

deleuon analym 155 electrophorest., 1.:19-50. 153-5 gel-shift analysis 156- ~ immunologtcal methods 169 mtcrococc:al nuclea'e dige~tion IM-5 ohgonudeotide-Rl\ase H targ•~ting 165-8 rate zonal centnfugation 15---60 clea~agc of pre-rRNA by nucleolar endonuclease 155 pre-rR!':A substrate. preparauon 144-8 proc-cssmg e\tracts preparation 148-53 processmg of pre-rRI'
~eH->phcmg

group lmtrons 211-15 group II introns 218-22 see also riboLymes splicing. see ~plicing substrate\ for processing reactio1u capping ol mR~A 38~ \\ilh defined termini 236- ~ editing mitochondrial mR:>IA s~-8 end-processing of mRNA 2-4, 15-16. 23-5 group I introns 215-r: group II introns 221-~ hammerhead and hairpin ribozyme< 233-5 pre-rRNA 144-8 pre·tRNA 177-85 turnOH'r of mRNA 109-11, 119-26 see also \' olume I targeting b~ hammerhead and hairpm nbozymes 232-6 RNase H 165-9 triphosphata\e 147 RNA-protein mteracuom in rR:'\1\ processing analysis by gel-shift 156-7 rate zonal centrifugauon 151-60 UV cross-lin kmg 160--1 RNase A, identifkauon ol tR:-.1.\ .plice ,jte~ 203-6 R:--lase, cryptic, 10 RNA editinF 96, 100-1 R:-iase H, for targeted destructton of RNA 165-9, see olso \'olume I RNase inhibitor~. in mR:>i-\ turno\er 110-11, 113-14 RNa~e protection. analysis of polynden) Jauon complexes 10-1-1 Ri'ia,e Tl anaJy,j, of polyadenylation compleses 13 identification of tR:-.1·\ splice 'lte< 203-6 RNa,e T2 analysts of mRNA cap> 52, 5-1-5, 59-60. 63-4 analysis of polyadenylation .:omplexe> D RI\'P complexes, anal)si' b\ electron micro-;cop~ 1-13-4 gel-~hift assay 156-7

252

Index tmmunological method~ 169 14.:!-3 rate ronal centrifugation 15i-60 L \ cro"-1inking 160-4 see also Volume I run-off transcription, see 111 1'/lro tran,cnption

terminal uridvlll transtcra~e 3>,J\' 97-9- in R~" '\ edit;ng 96-TLC of capped oligonucleoudes 61-2 tRNA nucleotides 201-4 trans-acting mRNA stabilit~ regulatory factor effect on mRNA turnover 129-30 m po~t-pol}somal supernatants 112-13 trans-a<"ting proce.>sing factors in 3 end-proces>ing of mR:-
p~oralcn cros~-linking

xhizoJeme typing of kinetopla~tid protozoa -:"8-9 SDS-PAGE. of ~nRNP protein, 162-4, see also Volume I sclt -splicing intron;, lrans-e~terification detection in cellular R:-JA 213-15 group I in R!\A editing 69-70, 90, 96, 101-2 detection 213-15 10 ~df-~plicing inLron~ 211-13,218-20, 2.!4 lramfer RNA mechanism 211-13 occurrence 211 ba!>e modificatiom 199-206 group II labelling in cell~ 178-79 proce,~ing ba;,ic reaction 222-3 a~sa) 195-9 di;,tmction from group 1 218 mechani'm 21S-19 endonuch.•a'e 189-91 modification;, 224 e\tract preparation 186-9 liga;.e 191 -5 transcnpts for 221-3 site-specific labelling of caps O\Crview of path\\ays 175-6 chemical 48-52 pre-tRNA wbstrates, prepararion 177-9, 181-8 b}· decappmg and recapping 45-1! 0\Cf'ie\\ 38-9, 45 purificarion ol factors 189-91 b) trans..:npuon 39-~ 'Piice site Identification 199-206 splicing 175. 186, 189, 196-9 snake \enom phosphodi~tera,e, in idenuficalion of tRNA splice >ite.> 201-3 tran~pOSIIIOn, see re\'Cf\e "Piicing of group 11 mtrons 'nRNA /raiiS-~plicing of group II introm 217-8 analysis by Northern blotUng 157, 160 rurno,er of mRNA m e\lracts clea,age b) RNase H 165-6. 168 anal) ,js 126-30 .:ro>s-lmking to pre-rR."'A 142-3 choice of sub,trale 108-10 in pre-rR"'A proccssmg 136. 142 1e£' also Volume 1 data inrerpretauon 130-1 of deadenylatcd mRNA 128 SP6 RNA polymera~c. !>ee bacteriophage deca~ rate~ 130-1 RNA pOI>merase, 111 1•itro transcription spheropla!>ts, yeast 187-8 detecuon of mR'IA 126-31 splicmg using endogenous mR\tA 119-21. 124 e\amples 126-JO of group I intron;, 211-17 of group II mtroru 21~-30 using exogenous mRNA 121-6 reverse splicmg 226-8 experimemal advantage\ I 07-R spliced exon reopemng 225-7 extract preparation 111-19 splice junction hydrolysis 225-7 inhibition of non-specific RNasc 113-14 splice junctions in tRNA 201-6 poi)(Al-binding protein, effect of 128-30 of tRNA 175. 186, 189, 196-9 preparation of undegraded mR!\A 110-11 problem ~ohing 131-2 !>£'£' also group I inrron ribozymes_ group 11 quanufication 126-31 Intron riboZ)me;,, and Volume 1 regulation 129-30, 132 stability factor for mRNA. see rrans-ac:ting two-dimensional chromalography of mRNA >lability regulatory factor capped oligonucleotide 6-1-5 tRNA nucleoude, 205-6

TJ, Ti R!'IA polymera-.e. see bacteriophage R:-J'\ polymerase. 111 \'itro tran•cription

L \ cro,\-Jinking of rR ~.'\-protein method 162--l

253

Index UV cross-linking of rRNA-protein (cont.) preparation of substituted rRNA 162-3 synthesis of 4-S-UTP 160-2

\iral transcription of capped Rl\A

38-43

Xenopus oocytes, pre-rRNA

proce~sing

in

168-70 yeast, RNA processing m group II imron ribozymes 217, 221. 125 tRNA processing 173-81, 183. 186-91. 194, 196-8, 201 see also Volume 1

254

ORDER OTHER TITLES OF INTEREST TODAY Price list for: UK, Europe, Rest of World (excluding US and Canada)

13 8. Pla•mitls (2 e) llord1 >. C: (Ed! £30.00 Sp~ralbound hardback 0·19·963445·9 £19.50 Paperback 0·19·963444·0 136. KNA Processing: Vol. II Hkgins. S I & Hdmcs . I! I) (Edsl Sp~ralbound hardback 0·19·963471·8 £30.00 0·19-963470-X £19.50 Paperback 135 Kt\A Processing: \ol. I lliAAtllS. !:>I !.. llomcs.

117

116 115

H.U llods)

0 19·963344-4 £30 00 Paperback 0-19·963343-6 £19 50 II.MK of Macromolecules Roberts. GC.>.. (Ed Sp~ralbound hardback 0·19·963225·1 £32 50 Paperback 0-19 963224·3 £22 50 Gas Chromatography B•uKh. I' {Ld) 133. Sp~ralbound hardback 0-19-963272-3 £40.00 £27.50 Paperback 0-19·963271 5 132. Essential Uevelopmenlal Hinlog} StNn (.ll & Holldnd 1'.II' H. (Eds) £30.00 Sp~ralbound hardback 0-19-963423·8 £19.50 Paperback 0·19-963422-X 131. Cellular Interaction~ in DeH•Iopment Hutlo·y n ..\ (Ed! Spiralbound hardback 0-19-963391·6 £30.00 Paperback D-19-963390·8 £18 50 129 Behavioural Neurosc..ienct>: \'olunw II s,,hgol. A (Ed) Sptralbound hardback 0-19·963458·0 £32.50 Paperback 0·19·963457-2 £22.50 128 Behavioural Neurnsc.ienct>: Volumr I Sah)owl. A.ll\<1) Spiralbound hardback 0·19·963388·1 £32.50 Paperback 0·19·963367-3 £22 50 127. Molel.ular Virolog} n.n·io::nn \ .I & Elhutt KM {Eels! Sporalbound hardback D-19·9633!'>8-4 £35.00 Paperback 0·19-963357-6 £25 00 12 6. GenP T argeting )nynrr, \ 1.. (Ed! Sp~ralbound hardback 0·19·963407·6 £30 00 Paperback 0·19·9634036-8 19.50 12 5. Gl) cobiolog) Fukuda. \f & >.ul>dl.t. \ lf.ds1 Sp~ralbound hardback 0·19-963372-X £32 50 Paperback 0·19·963371-1 £22.50 124. Human Genetic Disease Analysi' (2ie) Davips. >..E. tErti Sp~ralbound hardback 0-19-963309·6 £30 00 Paperback 0-19-963308-8 £18.50 122. fmmunocvtochemistn· nccslcv. 1- (l:'d) Sp~ralbound hardback · 0-19·963270· 7 £35 00 Paperback D-19-963269·3 £22.50 123. Protein Pho>phorylahon lidTdir• n (, iF.d Sp;ralbound hardback 0-19·963306-1 £32 50 Paperback 0-19-963305·3 £22 50 121. rumour lmmunobiolog) G
hardback

114. 113

112.

Geno Transtription HdiHt·~ H.U. & HiAAin"> S ){Eels) Sptralbound hardback 0-19963292-8 £35 00 Paperback D-19·963291 -X £25 00 F.lrctroph••siolog\ \\alias. U I (f:cll £32.50 Sp~ralbound hardback D-19-963348· 7 Paperback D-19-963347-9 £2250 lltolo~acal Uata Anal)>i> Frv I C ~ ll'd) Sptr a Ibound hardback D-19-963340-1 £50 00 Paperback D-19·963339-8 £27.50 t.x~rimenti! l '-curoanatom~

Oolam I.P I Ed

Sptralbound hardback 0·19·963326-6 £32 50 Paperback 0·19·963325-8 £22.50 Prt•parali\c Centrifugation Hid.wuod U (l•:,l j sp;ralbound hardback 0-19-963208-1 £45.00 Paperback 0-19-963211 1 £25.00 Paperback 0-19-963099 2 £25.00

l.ipid Analysi., Hdmiltou ltl & H.unillon Shicla ll:.d>l Sp1ralbound hardback

Paperback 111.

Haemopoie:.i~

0-19-963098-4 0-19·963099-2

£35.00 £25.00

IP...td, '\ (,. & \1ulin1~1l'C (,

icd>t

110. 109. -._J08.

107

106.

105. 104.

103.

102 101. 100.

99

Sp1ralbound hardback 0·19-963366·5 £32 50 Paperback 0·19-963365-7 £22.50 Pollination t:r olng} J1,afni. \ Sp~ralbound hardback 0·19-963299-5 £32 50 Paperback 0·19·963298-7 £22.50 In Situ Hybridization \\'ilkinson. lJ(, (l:'u) Spiralbound hard back 0-19·963328·2 £30.00 Paperback 0-19·963327-4 £18.50 Protein ~: ngineering RCL'S. A lt. stt~rnb.. rg. ~I ).F ~ otlll'rs iJ:tl,l Sparalbound hardbac• D-19·963139·5 £35.00 Paperback 0-19-963138·7 £25 00 Cell-Cell Inte rac tions Ste•·en"'"• ll R (,;allan. I\.) & uth~" IEtlsl Sparalbound hardback 0·19·963319-3 £32.50 Paperback 0-19-963318·5 £22.50 Diagnostir Molrc ular Patholog): Volume I Hrrrin~on, C.S. & ~Iductiun \lillig.m. G {Edt 0·19·963296·0 £30.00 Sparalbound hardback Paperback 0-19·963295-2 £18.50 Protein Targeting Magee, A .I & Wileman. T )Erbi Sparalbound hardback 0-19·963206-5 £32.50 Paperback 0·19·963210·3 £22.50 Diagnostic Molecular Pathology: Volume II: Cell and Tis~uP Genotyping llerrinHtun. C.S. & \.ILG<>e. I O'U !Eds) £30.00 0-19-963239-1 Spaalbound hardback £19.50 0-19-963238-3 Paperback Neuronal ~11 Lin~ \\'nod , I~ !Edt £32.50 Spualbound hardback D-19-963346·0 £22.50 Paperback D-19-963345·2

®

The Practical Approach Series SERIES EDITORS: D. RICKWOOD and B. D. HAMES In recent years, the importance of RNA processing in the regulation of eukaryotic gene expression has become abundantly clear and so, not surprisingly, it is now an area of intense research activity. These two volumes give detailed practical guidance on all major aspects of this subject. Step-by-step practical protocols from leading laboratories are presented for studies of the termination and end-processing, capping, methylation, splicing, and editing of mRNA as well as studies of mRNA stability and processing of rRNA and tRNA. The reader is led through all the key steps required for a successful experimental investigation. This includes full descriptions of the synthesis and purification of RNA substrates for in vitro work, the characterization of specific RNAs , and the isolation and analysis of ribonucleoprotein complexes.

RNA Processing: A Practical Approach. Volumes I and II, are invaluable laboratory companions for researchers working on mRNA, rRNA. or tRNA gene expression. Their lucid explanatory style and comprehensive coverage of all the key techniques for analysing RNA processing will be welcomed by both experienced researchers and workers embarking on such studies for the first time. Volume I 0 19 963344 4 spiral hardback 0 19 963343 6 paperback Two volume set 0 19 963473 4 spiral hardback 0 19 963472 6 paperback

ISBN 0-19-963470-X

OIRLPRESS

----at---

OXFORD UNIVERSITY PRESS Oxford New York Tokyo