Preface xv

Part One mRNA Biology 1

1 Introduction 3

1.1 RNA Building Blocks 4

1.2 RNA Folding 6

1.3 The RNA World Hypothesis 10

1.4 Functions of RNA 11

1.5 Protein Classes that are Required for RNA Function 12

1.5.1 RNA Binding Proteins 12

1.5.1.1 Proteins that Interact with Single Stranded RNAs 12

1.5.1.2 Proteins that Interact with Double Stranded RNAs 14

1.5.2 RNA Helicases 14

References 15

2 Transcription of Pre-mRNAs 17

2.1 Structure and Organization of Protein Coding Genes 18

2.2 Transcription of Mrnas by Rna Polymerase II 20

2.2.1 Transcriptional Initiation of Protein Coding Genes 23

2.2.2 Regulation of Transcriptional Initiation of Rna Polymerase II 27

2.2.3 Transition from Preinitiation to Initiation and Promoter Clearance 27

2.2.4 Productive Elongation of mRNA Transcripts 29

2.2.4.1 The Nucleotide Addition Cycle (NAC) 29

2.2.4.2 Protein Factors that Influence Pol II Elongation 30

2.3 Transcriptional Termination of Pre-mRNAs 31

2.4 Transcription is Coupled to Other mRNA Maturation Steps 32

2.5 Summary 34

References 35

3 Capping of the Pre-mRNA 5 0 End 37

3.1 m 7 G-cap Structure 37

3.2 mRNA Capping Enzymes 39

3.2.1 RNA Triphosphatase 39

3.2.2 Guanylyltransferase 39

3.2.3 Guanine-N7-Methyltransferase 40

3.3 5 0 Capping is Coupled to Transcription 41

3.4 5 0 Cap Binding Proteins 41

3.5 Summary 42

References 43

4 3 0 End Processing of Pre-mRNAs 45

4.1 Polyadenylation Signals 46

4.2 Proteins Involved in 3 0 End Processing of Pre-mRNAs 47

4.2.1 Cleavage and Polyadenylation Specific Factor (CPSF) 47

4.2.2 Cleavage Stimulation Factor (CstF) 47

4.2.3 Mammalian Cleavage Factor I (CFI m) and II (CFII m) 48

4.2.4 The Poly(A) Polymerase (PAP) 49

4.2.5 Poly(A)-Binding Protein (PABP) 50

4.2.6 Symplekin 51

4.3 3 0 End Processing is Tightly Linked to Transcriptional Termination 51

4.4 Alternative Polyadenylation 51

4.5 Cytoplasmic Polyadenylation 53

4.6 3 0 End Processing of Histone mRNAs 54

4.7 Summary 56

References 57

5 Splicing of Eukaryotic Pre-mRNAs 59

5.1 Group I, II and III Introns 59

5.1.1 Group I Introns 59

5.1.2 Group II Introns 61

5.1.3 Group III Introns 61

5.2 The Mechanism of pre-mRNA Splicing 61

5.3 The Spliceosome 62

5.4 The U12-Dependent Minor Spliceosome 66

5.5 Coupling of Splicing with Transcription and 5 0 Capping 67

5.6 Alternative Splicing and the Complexity of Genomes 68

5.6.1 Mechanisms of Exon Inclusion into the Mature mRNA 69

5.6.2 Mechanism of Exon Exclusion from the Mature mRNA 70

5.7 Summary 70

5.8 Questions 71

References 72

6 mRNA Export from the Nucleus to the Cytoplasm 73

6.1 Nuclear Import and Nuclear Export 73

6.2 mRNA Export Receptors 75

6.3 Adaptors that Bridge mRNAs with Export Receptors 78

6.4 Mechanism of mRNA Export 78

6.5 Coupling of mRNP Export to Other Steps of mRNA Maturation 80

6.6 Summary 80

6.7 Questions 81

References 81

7 Translation 83

7.1 Amino Acids, mRNAs, tRNAs 83

7.1.1 mRNA 83

7.1.2 Amino Acids 84

7.1.3 Transfer RNAs (tRNAs) 86

7.1.4 Loading Amino Acids onto tRNAs 87

7.2 The Ribosome 89

7.3 The Mechanisms of Translation 90

7.3.1 Translation Initiation 90

7.3.1.1 Eukaryotes 90

7.3.1.2 Bacteria 93

7.3.1.3 Archaea 96

7.3.1.4 Internal Ribosome Entry Sites (IRESs) 96

7.3.2 Elongation 97

7.3.2.1 Polyribosomes 97

7.3.3 Termination 99

7.3.4 Recycling of the Ribosome 100

7.4 Translational Regulation 100

7.4.1 Regulation of Translation Initiation 100

7.4.2 Regulation of Translation Elongation and Termination 103

7.5 Coupling Translation with Other mRNA Maturation and Quality Control Steps 103

7.6 Summary 104

7.7 Questions 105

References 106

8 Deadenylation of mRNA 107

8.1 Deadenylating Enzymes 107

8.1.1 Poly(A) Nuclease 107

8.1.2 CCR4-NOT Complex 108

8.1.3 Poly(A) Ribonuclease 108

8.1.4 Other Deadenylases 110

8.2 Summary 111

8.3 Questions 111

References 112

9 mRNA Decapping 113

9.1 Decapping Enzymes are the Core of the mRNA Decapping Machinery 113

9.2 Scavenger Decapping Enzyme DcpS 115

9.3 Regulation of mRNA Decapping 115

9.3.1 Inhibitors of Decapping 115

9.3.2 Enhancers of Decapping 116

9.4 Intracellular Localization of mRNA Decapping 117

9.5 Summary 118

9.6 Questions 119

References 119

10 mRNA Decay Pathways 121

10.1 Deadenylation-Dependent mRNA Decay 122

10.1.1 The 5 0 to 3 0 Exoribonuclease Xrn 1 122

10.1.2 The Exosome 122

10.1.2.1 Structural Organization of the Exosome 125

10.1.2.2 Mechanism of Exosome-Mediated RNA Degradation 125

10.1.2.3 Regulation of Exosome Activity 126

10.2 Deadenylation-Independent mRNA Decay 127

10.3 Endoribonuclease-Mediated mRNA Decay 128

10.3.1 Eukaryotic Endoribonucleases 129

10.4 Regulation of mRNA Decay 131

10.5 RNA Degradation in Bacteria 131

10.6 Summary 133

10.7 Questions 134

References 135

11 mRNA Quality Control 137

11.1 Nuclear mRNA Quality Control Mechanisms 137

11.1.1 MRNP Retention at the Transcription Site 138

11.1.2 MRNP Quality Control at the Nuclear Pore Complex 138

11.2 Nonsense-Mediated mRNA Decay (NMD) 138

11.2.1 Protein Factors Required for NMD 139

11.2.1.1 UPF Proteins 139

11.2.1.2 The Exon–Exon–Junction Complex (EJC) and Nmd 140

11.2.1.3 SMG Proteins and the Phosphorylation of UPF 1 141

11.2.2 Mechanism of NMD in Mammals 142

11.2.3 Cytoplasmic Processing Bodies and NMD 143

11.2.4 Mechanism of NMD in Yeast and Flies 144

11.2.5 mRNA Degradation Pathways in NMD 146

11.3 Other mRNA Quality Control Pathways 146

11.3.1 Non-Stop mRNA Degradation 146

11.3.2 No-Go mRNA Decay (NGD) 148

11.4 Summary 148

11.5 Questions 149

References 149

Part Two Non-Coding RNA Biology 151

12 Ribosomal RNAs and the Biogenesis of Ribosomes 153

12.1 Genomic Organization of Ribosomal RNA Genes 153

12.1.1 Bacteria and Archaea 153

12.1.2 Eukaryotes 155

12.1.2.1 28S, 18S and
5.8S rRNAs 155

12.1.2.2 5S rRNA 156

12.2 Transcription of Ribosomal RNA Genes 157

12.2.1 RNA Polymerase I 157

12.2.1.1 Initiation of Pol I Transcription 158

12.2.1.2 Promoter Clearance, Transcript Elongation and Termination of Pol I Transcription 160

12.2.1.3 Regulation of RNA Polymerase I Transcription 161

12.2.2 RNA Polymerase III and the Transcription of the 5S rRNA 162

12.2.2.1 Pol III Promoters 163

12.2.2.2 Transcription Initiation and Elongation of Rna Polymerase III 165

12.2.2.3 Initiation of Type 3 Promoters 167

12.2.2.4 Termination and Re-Initiation 168

12.3 Maturation of rRNAs 169

12.3.1 Small Nucleolar RNAs are Required for Pre-rRNA Processing 170

12.4 Assembly of Ribosomal Subunits 172

12.5 Nuclear Export of Ribosomal Subunits 174

12.6 Modification, Structure and Function of rRNAs 175

12.7 Summary 178

12.8 Questions 179

References 180

13 Transfer RNAs 183

13.1 Genomic Organization and Transcription of tRNA Genes 183

13.2 Processing to Mature tRNAs 184

13.2.1 5 0 Maturation of tRNAs by the RNase P Enzyme Complex 184

13.2.2 3 0 End Maturation of tRNAs 186

13.2.3 tRNA Splicing 188

13.3 tRNA Modifications 191

13.4 Nuclear Export of tRNAs 193

13.5 Tertiary Structure of tRNAs 194

13.6 Summary 196

References 197

14 The 7SL RNA and the Signal Recognition Particle 199

14.1 Architecture of the SRP 199

14.1.1 The SRP RNA 199

14.1.2 Protein Components of the SRP 201

14.1.2.1 Eukaryotes 201

14.1.2.2 Archaea and Bacteria 203

14.2 SRP-Mediated Protein Translocation 204

14.3 Summary 206

References 208

15 Regulation of Transcription: the 7SK Small Nuclear RNA 209

15.1 Architecture of the 7SK snRNA 209

15.1.1 The 7SK snRNA 209

15.1.2 Protein Components of the 7SK snRNP 210

15.2 The 7SK snRNP Functions as Transcriptional Regulator 212

15.2.1 P-TEFb Function in Transcription 212

15.2.2 Repression of P-TEFb by the 7SK snRNP 214

15.3 Other Small Non-Coding RNAs that Interfere with Transcription 214

15.3.1 The 6S RNA in Bacteria 214

15.3.2 Alu, B1 and B2 Non-Coding RNAs in Mammals 215

15.4 Summary 215

References 216

16 Small Nucleolar RNAs 217

16.1 Genomic Organization and snoRNA Transcription 217

16.2 Box H/ACA snoRNAs 218

16.3 Box C/D snoRNAs 221

16.4 Maturation of Functional snoRNPs 223

16.5 Orphan snoRNAs 224

16.6 The Telomerase RNP 226

16.7 Summary 227

References 228

17 Spliceosomal Small Nuclear RNAs 229

17.1 Transcription and Maturation of Spliceosomal snRNAs 229

17.1.1 Transcription of Spliceosomal snRNAs 229

17.1.2 snRNA Maturation 230

17.1.3 SnRNA Export to the Cytoplasm 231

17.2 The Structure of UsnRNPs 232

17.2.1 Secondary Structure of Spliceosomal snRNAs 232

17.2.2 Protein Composition of UsnRNPs 233

17.2.2.1 The Sm/LSm Core Structure 233

17.2.2.2 UsnRNP-Specific Proteins 235

17.3 Assembly of Spliceosomal snRNPs 237

17.3.1 Cytoplasmic Assembly of the Sm Core Domain 237

17.3.2 Formation of the Tri-Methyl Guanine Cap 240

17.3.3 Import of Assembled UsnRNPs into the Nucleus 241

17.4 Summary 242

17.5 Questions 243

References 244

18 Small Non-Coding RNAs and the Mechanism of Gene Silencing 245

18.1 Short Interfering RNAs and the Mechanism of RNA Interference 245

18.2 Dicer 248

18.3 RNA-Dependent RNA Polymerases 248

18.4 Argonaute Proteins 251

18.5 microRNAs and the Regulation of Gene Expression 251

18.5.1 MiRNA Biogenesis 251

18.5.2 Non-Canonical miRNA Biogenesis Pathways 253

18.5.3 miRNA Functions 255

18.5.3.1 miRNAs Can Act as siRNAs 255

18.5.3.2 miRNAs Inhibit Translation 255

18.5.3.3 miRNAs Induce Deadenylation and mRNA Decay 256

18.6 PiRNAs and the Regulation of Mobile Genetic Elements in the Germ Line 257

18.6.1 Transposons as Driving Force Behind Evolution 258

18.6.2 PiRNAs Control Transposon Expression 259

18.7 Small RNAs with Functions in Chromatin Regulation 261

18.8 The CRISPR System – A Bacterial and Archaeal Defense Mechanism 263

18.8.1 The CRISPR Locus 263

18.8.2 Acquisition of CRISPR-Mediated Resistance 264

18.8.3 Mechanism of CRISPR Activity 265

18.9 Summary 266

References 269

19 Long Non-Coding RNAs 271

19.1 The XIST Non-Coding RNA and X Chromosome Inactivation 271

19.1.1 The X-Chromosome Inactivation Center (XIC) 272

19.1.2 the Xist Non-coding Rna and the Mechanism of X Inactivation 272

19.1.3 Regulation of XIST Function 274

19.2 Dosage Compensation in Flies 275

19.3 Non-Coding RNAs and the Regulation of Imprinting 276

19.4 The Regulation of HOX Genes by Long Non-Coding RNAs 278

19.5 Long non-Coding RNAs are Common in Complex Genomes 278

19.6 Summary 278

References 280

20 RNA Editing 281

20.1 RNA Editing by U Insertions or Deletions 281

20.1.1 Mechanisms of U Insertions or Deletions 282

20.2 RNA Editing by Base Modification 283

20.2.1 c to U conversion 284

20.2.2 Adenine to Inosine Editing 286

20.2.2.1 Adenosine Deaminase Acting on RNA 287

20.2.2.2 Editing Site Selectivity 288

20.2.2.3 Biological Consequences of A to I Conversions 288

20.3 Summary 290

References 291

21 Ribozymes – Catalytic RNA Molecules 293

21.1 Identification of Catalytic RNAs 293

21.2 Mechanisms and Secondary Structures of Different Ribozymes 294

21.2.1 Group I Introns 294

21.2.2 RNase P 295

21.2.3 The Diels–Alderase Ribozyme 296

21.2.4 Hammerhead Ribozymes 298

21.2.5 The glmS Ribozyme 298

21.3 Summary 300

References 301

22 Riboswitches and RNA Sensors 303

22.1 Mechanisms of Riboswitch Function 303

22.2 Riboswitch Structures 305

22.3 RNA Thermometers 305

22.4 Summary 307

References 308

23 RNomics 309

23.1 ‘‘Omics’’ Approaches 309

23.2 Experimental RNA Profiling Strategies 310

23.2.1 Northern Blotting 310

23.2.2 Microarray 311

23.2.3 Quantitative PCR 313

23.2.4 RNA Fluorescent In Situ Hybridization 314

23.2.5 Next Generation Sequencing 314

23.3 RNA Biology and the Complexity of Genomes 315

23.4 Summary 315

References 318

Appendix: Answers to Questions 319

Index 355