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
1.5.2 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
2.2.4 Productive Elongation of mRNA Transcripts ....... 29
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' End .............................. 37
3.1 m7G-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' Capping is Coupled to Transcription ................. 41
3.4 5' Cap Binding Proteins ................................ 41
3.5 Summary ................................................ 42
References .................................................. 43
4 3' End Processing of Pre-mRNAs .............................. 45
4.1 Polyadenylation Signals ................................ 46
4.2 Proteins Involved in 3' 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 (CFIm) and II
(CFIIm) ......................................... 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' End Processing is Tightly Linked to
Transcriptional Termination ............................ 51
4.4 Alternative Polyadenylation ............................ 51
4.5 Cytoplasmic Polyadenylation ............................ 53
4.6 3' 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'
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
7.3.1 Translation Initiation .......................... 90
7.3.2 Elongation ...................................... 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' to 3' Exoribonuclease Xrnl .............. 122
10.1.2 The Exosome .................................... 122
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
11.2.2 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
12.2 Transcription of Ribosomal RNA Genes .................. 157
12.2.1 RNA Polymerase I ............................... 157
12.2.2 RNA Polymerase III and the Transcription of
the 5S rRNA .................................... 162
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' Maturation of tRNAs by the RNase P Enzyme
Complex ........................................ 184
13.2.2 3' 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
14.2 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
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
17.3 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
18.6 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 С to U Conversion .............................. 284
20.2.2 Adenine to Inosine Editing ..................... 286
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
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