RNA:Protein Interactions

RNA:Protein Interactions

List of contributors Abbreviations 1. Applications of chemically synthesized RNA Michael J. Gait, David J. Earnshaw, Mark A. Farrow, Jan H. Fogg, Richard L. Grenfell, Nikolai A. Naryshkin, and Terence V. Smith 1. Basic methods of synthesis and purification of oligoribonucleotides Introduction Assembly of oligoribonucleotides Deprotection of oligoribonucleotides Purification of oligoribonucleotides 2. Incorporation of modified ribonucleotides Base analogues Ribose analogues Phosphate analogues 3. Analysis of oligoribonucleotides and analogues Capillary electrophoresis MALDI-TOF mass spectrometry Analysis of oligoribonucleotides by enzymatic digestion and reversed-phase HPLC 4. Synthetic RNA duplex models for HIV-1 Tat protein interaction Competition gel retardation assays Interference filter binding assays 5. Cross-linking of peptides and proteins to modified synthetic RNA Acknowledgements References 1 1 2 5 8 12 12 15 16 18 18 20 21 23 23 27 29 33 33 2. Preparation of RNA:protein complexes for X-ray crystallography and NMR 37 Stephen R. Price, Chris Oubridge, Gabriele Varani, and Kiyoshi Nagai 1. Introduction 37

2. Preparation of T7 RNA polymerase 38 3. In vitro transcription using oligonucleotide or plasmid templates 40 RNA synthesis by run-off transcription using oligonucleotide templates 40 In vitro transcription using plasmid templates 43 4. Gel purification of transcribed RNA 51 Casting gels for preparative RNA purification 52 Extraction of RNA from polyacrylamide gels 53 Desalting RNA 55 Concentration of RNA 58 5. Preparation of isotopically labelled RNA for NMR 58 Purification of isotopically labelled RNA from bacterial sources 59 6. Preparation of RNA:protein complexes for crystallization and NMR analysis 66 Checking purity of RNA 66 Reconstituting the RNA:protein complex 69 Crystallization strategy of RNA:protein complexes 71 Acknowledgements 72 References 72 3. Uses of site-specifically modified RNAs constructed by RNA ligation 75 Melissa J. Moore and Charles C. Query 1. Introduction 75 _ 2. Review of methods for introducing modified nucleotides into small RNAs 75 Chemical synthesis 77 Transcription with phage polymerases 77 Coupling chemical synthesis to transcription: dinucleotide primers 78 3. Synthesis of long RNAs by transcription 78 4. Techniques for RNA ligation T4 RNA ligase T4 DNA ligase Chemical ligation 5. Examples of cross-linking proteins to site-specifically modified RNAs Photocross-linking proteins to RNA Patch labelling Using T4 DNA ligase to incorporate single labels at internal sites in RNAs xii

Convertible nucleotides 98 Other photocross-linkable nucleotides 102 Determining cross-link specificity 103 6. Additional uses for RNA ligation 105 References 106 4. The electrophoretic mobility shift assay for RNA binding proteins 109 Douglas L. Black, Raymond Chan, Hosung Min, Jiwu Wang, and Leslie Bell 1. Introduction 109 Principle of electrophoretic mobility shift assay (EMSA) 109 General considerations 109 2. Starting material: RNA and protein 111 Preparation of mammalian nuclear extracts 111 Preparing radiolabelled RNA for binding studies 113 Large scale synthesis of unlabelled RNA 114 3. EMSA for protein:rna interactions 114 Binding conditions for the DCS complex " 114 Separation of RNA:protein complexes by native gel electrophoresis 115 Optimizing the binding reaction 116 The stability of an RNA:protein complex within the gel 117 Measuring the sequence specificity of binding 119 4. Characterization of the proteins within a complex 120 UV cross-linking within a gel isolated complex 120 Antibody supershift and disruption experiments 123 EMSA as an assay for fractionation 124 5. RNA binding analysis of purified proteins 124 EMSA with purified protein 124 Filter binding assay 125 Establishing the binding conditions 126 Determining the dissociation constant 128 Multiple binding sites and co-operativity 130 References 135 5. Affinity methods for isolating RNA binding proteins 137 Ann Kaminski, Dirk H. Ostareck, Nancy M. Standart, and Richard J. Jackson 1. Introduction 137 Scope of the chapter 137 xiii

Overview of methods for affinity purification of RNA binding, proteins 138 Protein purification prior to affinity chromatography 139 Suggestions with regard to prioritizing approaches 140 2. Large scale transcription reactions using bacteriophage RNA polymerases 141 3. General recommendations for running RNA affinity columns 143 4. Coupling RNA to CNBr-activated Sepharose 144 Use of affinity columns made by coupling RNA to CNBr-activated Sepharose 146 5. The use of poly(a) tailed RNA affinity substrates 148 6. Biotinylated RNA as an affinity matrix 150 Overview of methods for preparing biotinylated RNA affinity substrates 150 Modification of RNA by direct incorporation of biotinylated NTP 151 Two-step modification of RNA by incorporation of an amine-modified NTP, and subsequent biotinylation with biotinylated ester 153 Synthetic biotinylated oligoribonucleotides 154 Recovery of RNA binding proteins from the biotinylated RNA affinity matrix 155 7. Other affinity methods for purifying RNA binding proteins 156 8. The use of affinity columns for selectively removing specific RNA binding proteins from extracts 157 Acknowledgements 158 References 158 6. Synthetic lethal/enhancer screening to identify snrna:protein and protein:protein interactions in yeast pre-mrna splicing i6i Francoise Stutz, Jie Tang, and Michael Rosbash 1. Introduction 161 2. Ul snrna enhancer screen using a viability assay 165 General reagents and strains > 165 Protocols 166 Results and discussion of Ul snrna enhancer screen 171 3. Identification of synthetic lethal mutations using the red/white sectoring assay 175 General reagents and strains 176 xiv

Protocols 177 Results and discussion of MUD2 synthetic lethal screen 180 4. Concluding remarks 181 References 181 7. Detecting RNA:protein interactions and isolating cdna clones by Northwestern screening 183 Jeffrey Wilusz 1. Introduction 183 General considerations for Northwestern detection of RNA:protein interactions 183 Probability of success 184 2. Preparation of RNA probes 185 3. Northwestern blots to detect RNA binding proteins 187 4. Northwestern screening of cdna libraries 189 References 193 8. A three-hybrid system to detect and analyse RNA:protein interactions in vivo 195 Beilin Zhang, Brian Kraemer, Dhruba Sengupta, Stanley Fields, and Marvin Wickens 1. Introduction 195 2. Strategy of the three-hybrid system 196 3. General considerations 197 4. Potential applications 199 5. Hybrid RNA molecules: general features 200 Constructing plasmids that encode hybrid RNA molecules 201 6. Reporter strain 204 7. The activation domain plasmid 205 8. Analysing known RNA:proteiri interactions 205 9. Screening for new proteins with a known RNA sequence: general strategy and considerations 210 10. Perspective 215 Acknowledgements 215 References 215 xv

9. In vivo selection of specific RNA binding polypeptides using a transcription anti-termination reporter assay 217 Kazuo Harada and Alan D. Frankel 1. Introduction 217 2. Detection of RNA:polypeptide interactions using the bacterial transcription anti-termination system 218 3. Strategies in the design of combinatorial libraries 222 Library design 222 Preparation of combinatorial oligonucleotide cassettes 225 4. Selection of novel peptides that bind to specific RNAs 229 Selection from randomized libraries 229 Selection from doped libraries 231 5. Assessing activity 232 (3-Galactosidase solution assay 232 In vitro RNA binding gel shift assay 233 6. Summary and future directions 235 References 235 10. Footprinting and modification-interference analysis of binding sites on RNA 237 Chuck Merryman and Harry F. Noller 1. Introduction 237 2. Experimental approach 238 General considerations 238 Footprinting 238 Modification-interference 239 3. Chemical probing of RNA 240 Chemical probes 240 Probing conditions for footprinting experiments 241 Probing conditions for modification-interference experiments - 241 Protocols for chemical probing 242 4. Primer extension 246 5. Interpretation 249 Footprinting 249 Modification-interference 251 Concluding remarks 251 References 253 xvi

'Contents 11. Analysis of RNA:protein and RNA:RNA contacts between ribosomes and functional ligands using site-directed cross-linking techniques 255 Jutta Rinke-Appel and Richard Brimacombe 1. Introduction 255 2. General considerations 256 3. Insertion of photoreactive agents into ribosomal ligands 259 Preparation of mrna analogues containing 4-thioU by T7 polymerase 259 The use of diazirine or azide reagents for specific reaction with trna molecules at naturally modified positions 264 Modification of amino acids with TDB for in situ peptide biosynthesis 266 4. Formation of ribosomal complexes for cross-linking and isolation of cross-linked material 268 Preparation of 70S ribosomes 268 Separation of cross-linked ribosomal components by sucrose gradient centrifugation 272 Affinity chromatography on oligo(dt) cellulose 273 Cross-linking yield 274 5. Analysis of cross-link sites within rrna 274 Partial digestions of rrna by ribonuclease H in the presence of oligodeoxynucleotides 274 Identification of cross-link sites within rrna by primer extension 277 6. Identification of cross-linked proteins 277 7. Identification of cross-linked 4-thioU residues in mrna analogues 279 Identification of cross-linked 4-thioU residues from agarose-antibody complexes 279 Identification of cross-linked 4-thioU residues from ribonuclease H digests 281 Identification of 4-thioU residues cross-linked to ribosomal proteins separated after ribonuclease Tl digestion 281 8. Concluding remarks 282 References 283 12. In vitro selection of nucleic acid ligands 285 Richard C. Conrad, F. Maike Briick, Sabine Bell, and Andrew D. Ellington 1. Introduction 285 xvii

2. Overview 286 3. Creation of the initial pool 288 Design of the degenerate oligonucleotide population 289 Solid state DNA synthesis 290 Amplification to generate a dsdna library 292 Conversion of the initial dsdna library to different types of pools 297 4. Selection 301 Binding reaction 301 Methods for isolating target:aptamer complexes 304 5. Amplification" 310 6. Cycling and concluding the selection 313 7. Characterizing aptamers 315 Cloning, sequencing, and structure determination 315 Binding assays 317 Acknowledgements 323 References 324 Al. List of suppliers 327 Index 335 XV1U