Alternative pre-mRNA splicing: theory and protocols
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| Format: | Buch |
| Sprache: | English |
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Wiley-VCH
2012
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| Online-Zugang: | Inhaltstext Inhaltsverzeichnis |
| Beschreibung: | Auch angekündigt als: RNA splicing |
| Beschreibung: | XXXVII, 622 S. graph. Darst. |
| ISBN: | 9783527326068 |
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Datensatz im Suchindex
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Edited by
Stefan Stamm, Chris Smith, and Reinhard Luhrmann
Alternative pre-mRNA Splicing
Theory and Protocols
WILEY-BLACKWELL
Contents
Preface XXIII
List of Abbreviations XXV
List of Contributors XXXI
Part One Theory 1
1 Splicing in the RNA World 3
Emanuele Buratti, Maurizio Romano, and Francisco E Baralle
1 1 Introduction: The Fascination of Alternative Pre-mRNA Splicing 3
1 2 RNA Can Adopt a Flexible Conformation 4
1 3 Enzymatic RNAs and the RNA World 5
1 4 Common Classes of Eukaryotic RNA 6
1 5 Alternative Pre-mRNA Splicing as a Central Element of Gene Expression 8
1 6 Increasing Numbers of Human Diseases are Associated with Wrong
Splice Site Selection 9
References 11
2 RNPs, Small RNAs, and miRNAs 13
Michaela Beitzinger and Gunter Meister
2 1 Introduction 13
2 2 Ribonuclease P (RNase P) 14
2 3 Small Nucleolar RNAs (snoRNAs) 15
2 4 Small Regulatory RNAs 16
241 Short Interfering RNAs (siRNAs) 16
242 MicroRNAs (miRNAs) 17
243 Piwi-Interacting RNAs (piRNAs) 17
2 5 7SL RNA 17
2 6 7SKRNA 18
2 7 U-Rich Small Nuclear RNAs (U snRNAs) 18
References 18
3 RNA Elements Involved in Splicing 21
William F Mueller and KlemensJ Hertel
3 1 Introduction 21
3 2 Splice Site Sequence 22
3 3 Intron/Exon Architecture 23
3 4 Splicing Regulatory Elements (SREs) 24
3 5 RNA Secondary Structure 26
3 6 Coupling between Transcription and RNA Processing 27
3 7 Combinatorial Effects of Splicing Elements 28
References 29
VI Contents
4 A Structural Biology Perspective of Proteins Involved in
Splicing Regulation 33
Antoine Clery and Frederic H -T Allain
4 1 Introduction 33
4 2 The RRM: A Versatile Scaffold for Interacting with Multiple RNA
Sequences and also Proteins 35
421 RRM-RNA Interaction and Splicing Regulation 35
4211 RNA Binding by Splicing Factors Containing a Single RRM 36
4212 RNA Binding by Splicing Factors Containing Multiple RRMs 37
422 RRM-RRM and RRM-Protein Interactions in Splicing Regulation 41
4221 RRM-Protein Interactions Without RNA Binding 41
4222 RRM-Protein Interactions Allowing RNA Binding 41
4223 Impact of RRM-RRM Interactions on Splicing Mechanism 42
4 3 The Zinc Finger Domain 42
4 4 The KH Domain 44
4 5 Conclusions and Perspectives 45
References 46
5 The Spliceosome in Constitutive Splicing 49
Patrizia Fabrizio and Reinhard Luhrmann
5 1 Introduction 49
5 2 The Mechanism of Splicing 49
5 3 The Stepwise Assembly Pathway of the Spliceosome 51
5 4 Dynamics of the Spliceosomal RNA-RNA Rearrangements 53
5 5 Splice-Site Recognition and Pairing Involves the Coordinated
Action of RNA and Proteins 55
5 6 Driving Forces and Molecular Switches Required During the
Spliceosome's Activation and Catalysis 55
57A Conformational Two-State Model for
the Spliceosome's Catalytic Center 56
5 8 Compositional Dynamics and Complexity of the Spliceosome 57
5 9 Reconstitution of Both Steps of S cerevisiae Splicing with
Purified Spliceosomal Components 61
5 10 Evolutionarily Conserved Blueprint for
Yeast and Human Spliceosomes 61
5 11 Concluding Remarks 63
References 63
6 The Use of Saccharomyces cerevisiae to Study the Mechanism
of pre-mRNA Splicing 65
Brian C Rymond
6 1 Introduction 65
6 2 The Basics of Splicing 65
6 3 Yeast Intron-Exon Organization 66
6 4 The Yeast Spliceosome 67
6 5 Defining the Constellation of Yeast Splicing Factors:
Primary Screens and Genomic Inspection 68
6 6 Reporter Genes as Readouts of Splicing Efficiency 69
6 7 Genetic Interaction: Dosage Suppression or Antagonism 70
6 8 Extragenic Suppressors 70
6 9 Synthetic Lethality 71
6 10 Systematic Approaches to Define the Interactome 72
References 74
7 Challenges in Plant Alternative Splicing 79
Andrea Barta, Yamile Marquez, and John W S Brown
7 1 Introduction 79
Contents VII
7 2 Plant Introns 80
7 3 The Plant Spliceosome 81
7 4 Plant Spliceosomal Proteins 86
7 5 Alternative Splicing in Plants 88
References 90
8 Alternative Splice Site Selection 93
Miguel B Coelho and Christopher W J Smith
8 1 Introduction 93
8 2 The Players: Splicing Regulators 95
8 3 The Stage: The Splicing Complex Assembly and Exon Definition 96
8 4 Switching Splicing Patterns 99
8 5 Src Nl Exon: A Model of Combinatorial Splicing Regulation 101
8 6 The Global View: Towards a Splicing Code 103
References 104
9 Integration of Splicing with Nuclear and Cellular Events 109
Aparna K Sapra and Karla Neugebauer
9 1 Introduction 109
9 2 Overview 109
9 3 Nuclear Structure and Distribution of Splicing Factors 110
931 Cajal Bodies (CBs) 111
932 Splicing Factor Compartments (SFCs)/Speckles 111
933 Paraspeckles 111
9 4 Integration of Splicing with Nuclear and Cellular Processes 111
941 Splicing and Transcription 112
942 Splicing and mRNA Capping 112
943 Splicing and 3' End Processing 113
944 Splicing and Export 113
945 Splicing and Translation 114
946 Splicing and Nonsense-Mediated Decay (NMD) 114
947 Splicing and Chromatin Structure 115
References 115
10 Splicing and Disease 119
Emanuele Buratti and Francisco E Baralle
10 1 Introduction 119
10 2 Splicing and Disease 120
10 3 Therapeutic Approaches 121
10 4 The Generation of Aberrant Transcripts 122
10 5 Exon Skipping 122
10 6 Cryptic Splice Site Activation 122
10 7 Intron Retention 123
10 8 Pseudoexon Inclusion 124
10 9 Unexpected Splicing Outcomes Following the Disruption of Classical
Splicing Sequences 124
10 10 Conclusions 125
References 125
11 From Bedside to Bench: How to Analyze a Splicing Mutation 129
Marco Baralle and Diana Baralle
11 1 Introduction 129
11 2 From Clinical Evaluation to Mutation Testing 130
11 3 An Example of an Uncertain Diagnosis 132
11 4 Mutation Testing Procedures 133
11 4 1 In-Vitro Splicing 134
11 4 2 Minigene Splicing 134
VIII Contents
11 5 Concluding Remarks
References 137
12 Analysis of Common Splicing Problems 141
Stefan Stamm
12 1 Introduction 141
12 2 Is a Mutation Causing a Change in AS? 142
12 3 How is a Splicing Event Regulated, and How Can it be Influenced? 143
12 4 Is There a Difference in Alternative pre-mRNA Processing Between
Two Cell Populations? 144
References 145
13 Ultracentrifugation in the Analysis and Purification of Spliceosomes
Assembled In Vitro 147
Klaus Hartmuth, Maria A van Santen, and Reinhard Luhrmann
13 1 Theoretical Background 747
13 2 Protocol 148
13 2 1 Preparation of the Gradient 148
13 211 Manual Gradient Formation 148
13 212 Automatic Gradient Formation with the Gradient Master 149
13 2 2 Preparing the Run 150
13 221 Loading the Sample 150
13 222 Sedimentation Markers 150
13 2 3 The Ultracentrifuge Run 150
13 2 4 Harvesting the Gradient 150
13 3 Example Experiment 151
13 3 1 Purification of the Spliceosomal B Complex 151
13 311 Preliminaries 151
13 312 Preparation of the Spliceosomal B Complex 151
13 4 Troubleshooting 153
References 153
14 Chemical Synthesis of RNA 155
Claudia Hobartner
14 1 Theoretical Background 155
14 1 1 RNA Solid-Phase Synthesis 155
14 1 2 RNA Modifications 158
14 121 RNA Modification During Solid-Phase Synthesis 158
14 122 Post-Synthetic RNA Modification 158
14 1 3 Combined Chemical and Enzymatic Strategies 158
14 2 Representative Protocols 160
Protocol 1: Incorporation of Modified Phosphoramidites During
Solid-Phase Synthesis 160
Protocol 2: Coupling of Biophysical Probes to Aliphatic Amino
Groups on RNA 160
Protocol 3: Enzymatic Ligation of RNA fragments using
T4 RNA or T4 DNA Ligase 160
14 3 Troubleshooting 161
References 161
15 RNA Interference (siRNA, shRNA) 165
Daphne S Cabianca and Davide Cabellini
15 1 Theoretical Background 165
15 1 1 RNAi 165
15 1 2 siRNAs and shRNAs 165
Contents IX
15 1 3 Lentiviral-Mediated RNAi 167
15 2 Protocol 167
15 2 1 MapofpLKO l Puro 167
15 2 2 Oligonucleotide Design 167
15 221 Determining the Optimal 21-mer Targets in the Gene 167
15 222 Ordering Oligos Compatible with pLKO l 168
15 2 3 Generating the pLKO l Puro with a shRNA Construct 169
15 231 Annealing of the Oligonucleotides 169
15 232 Preparation of pLKO 1 TRC for Cloning 169
15 233 Ligating and Transforming into Bacteria 169
15 234 Screening for Inserts 169
15 2 4 Production of Lentiviral Particles 169
15 2 5 Lentiviral Infection 170
15 3 Example Experiment 171
15 4 Troubleshooting 172
References 172
16 Expression and Purification of Splicing Proteins 175
James Stevenin and Cyril F Bourgeois
16 1 Theoretical Background 175
16 2 Protocol 1: The Preparation of Total HeLa SR Proteins 175
16 2 1 Example Experiment 176
16 2 2 Troubleshooting and Important Points 177
16 3 Protocol 2: The Purification of Individual SR Proteins 177
16 3 1 Expression of SR Proteins in Escherichia coli and Purification 177
16 3 2 Preparation of SR Proteins Using a Baculovirus System 178
16 3 3 Example Experiment 178
16 3 4 Troubleshooting and Important Points 178
16 3 5 Production and Purification of Individual SR Proteins in Mammalian
Cells 179
References 179
17 Detection of RNA-Protein Complexes by Electrophoretic Mobility Shift
Assay 183
Manli Shen and Michael C Fried
17 1 Theoretical Background 183
17 1 1 Choice of RNA Substrate 184
17 1 2 Detection and Quantitation of Binding 184
17 1 3 Fluorescence 185
17 1 4 Chromogenic and Chemiluminescent Detection Methods 186
17 1 5 Stability of RNA-Protein Complexes During Electrophoresis 186
17 1 6 Competing Nucleic Acids and Polyanions 187
17 1 7 Binding Stoichiometry 187
17 1 8 Measurement of Binding Activity 187
17 1 9 Measurement of Dissociation Constants 188
17 1 10 Binding Competition 190
17 2 Protocol 190
17 2 1 Equipment 191
17 2 2 Reagents 191
17 2 3 Gel Preparation 191
17 2 4 Pre-Electrophoresis 192
17 2 5 Sample Preparation 192
17 2 6 Electrophoresis and Imaging 193
17 3 Example Experiment 194
17 A Troubleshooting 195
References 196
Contents
18 Functional Analysis of Large Exonic Sequences Through
Iterative In Vivo Selection 201
Ravindra N Singh and Natalia N Singh
18 1 Theoretical Background 201
18 1 1 Spinal Muscular Atrophy 202
18 2 Protocol 202
18 2 1 Minigene, Cell Culture, Transfection, and In Vivo Splicing
Assay 202
18 2 2 Generation of a Partially Random Exon 204
18 2 3 In vivo Selection 204
18 2 4 Analysis of Sequences 204
18 3 Example Experiment 205
18 3 1 Generating the Initial Pool of Splicing Cassettes 206
18 3 2 In Vivo Selection Procedure 207
18 4 Troubleshooting 209
References 209
19 Identification of Splicing c/'s-Elements Through an Ultra-Refined
Antisense Microwalk 211
Natalia N Singh, Joonbae Seo, and Ravindra N Singh
19 1 Theoretical Background 211
19 2 Protocol 212
19 2 1 Oligonucleotide Design and Synthesis 212
19 2 2 Cell Culture, Transfection, and In Vivo Splicing Analysis 212
19 2 3 Validation of Specificity 212
19 3 Example Experiment 214
19 4 Troubleshooting 216
References 217
20 Cenomic SELEX to Identify RNA Targets of Plant
RNA-Binding Proteins 219
Olga Bannikova, Maria Kalyna, and Andrea Barta
20 1 Introduction 219
20 2 Protocols 221
20 3 Example Experiment 225
20 4 Troubleshooting 225
References 226
21 Quantification of Alternative Splice Variants 229
Miriam Llorian and Christopher WJ Smith
21 1 Theoretical Background 229
21 1 1 PCR for Splice Variant Quantification 229
21 1 2 Conventional RT-PCR 230
21 1 3 Real-Time PCR: Chemistry and Quantification Methods 230
21 1 4 Primer Design 232
21 2 Protocol 23 3
21 2 1 RNA Extraction 233
21 2 2 DNase Treatment 233
21 2 3 Reverse Transcriptase Reaction 233
21 2 4 Radiolabeled PCR 233
21 2 5 qPCR Reactions 234
21 2 6 Data Analysis 234
21 3 Example Experiment: Microarray Validation of PTB-Regulated
Events 234
Contents XI
21 3 1 Primer Design 235
21 4 Troubleshooting 235
References 236
22 High-Throughput Analysis of Alternative Splicing by RT-PCR 239
Roscoe Klinck, Benoit Chabot, and Sherif Abou Elela
22 1 Theoretical Background 239
22 1 1 Endpoint PCR for the Detection of Alternative Splicing
Events 239
22 1 2 Computational Identification of Alternative Splicing Events 241
22 1 3 Primer Design 241
22 1 4 Capillary Electrophoresis 242
22 1 5 Data Analysis 242
22 1 6 Validation of Microarray and RNASeq Data 242
22 1 7 Tissue-Specific Annotation from Sequence Databases 243
22 2 Protocol 243
22 2 1 Primer Design 243
22 2 2 RNA Preparation 243
22 2 3 RTandQCofcDNA 244
22 2 4 PCR Reactions and Amplicon Detection 244
22 3 Example Experiment 245
22 4 Troubleshooting 245
References 246
23 Monitoring Changes in Plant Alternative Splicing Events 249
Craig C Simpson, Naeem Hasan Syed, Sujatha Manthri,
John D Fuller, Monika Maronova, Branislav Kusenda, Maria Kalyna,
Andrea Barta, and John W S Brown
23 1 Theoretical Background 249
23 1 1 Alternative Splicing in Plants 249
23 1 2 Splicing Analysis 250
23 1 3 Establishing the RT-PCR Conditions 250
23 1 4 Characterizing Novel Alternatively Spliced Products 250
23 1 5 Identifying AS Events in Genes of Interest 251
23 2 Protocols 251
23 3 Example Experiments 256
23 4 Troubleshooting 257
References 258
24 Array Analysis 261
Pierre de la Grange
24 1 Theoretical Background 261
24 1 1 Microarrays: General Principles 261
24 1 2 Probe Design of Splicing Microarrays: Interest and
Limitation 262
24 1 3 Available Splicing Microarrays 262
24 1 4 The Different Steps of the Microarray Data Treatment 263
IA2 Protocol 263
24 2 1 Normalization 264
1M2 Background Subtraction 264
24 2 3 Statistical Analysis 264
24 2 4 Visualization of Data 265
24 2 5 Functional Analysis of Results 265
24 3 Example Experiment 265
24 4 Troubleshooting 266
References 267
XII Contents
25 The CLIP Method to Study Protein-
RNA Interactions in Intact Cells and Tissues 269
James Tollervey andJernej Ule
25 1 Theoretical Background 269
25 2 Protocols 270
25 2 1 Material and Reagents 270
References 277
26 RNA-Protein Crosslinking and Immunoprecipitation (CLIP)
in Schizosaccharomyces pom be 281
Branislav Kusenda and Andrea Barta
26 1 Introduction 281
26 2 Protocol 282
26 2 1 Materials 282
26 2 2 Growth Conditions and Cell Harvest 283
26 2 3 Crosslinking 283
26 2 4 Sonication of the Cells 284
26 2 5 Immunoprecipitation 284
26 2 6 DNase Treatment 284
26 2 7 RNase Treatment 284
26 2 8 Dephosphorylation 284
26 2 9 L3 Linker Ligation 284
26 2 10 Phosphorylationofthe5' End of RNA 285
26 2 11 L5 Linker Ligation 285
26 2 12 Elution of the Protein-RNA Complex 285
26 2 13 cDNA Synthesis 285
26 2 14 PCR Amplification 285
26 2 15 Size Selection of DNA Fragments 286
26 2 16 Cloning Step: Generation of Overhangs 286
26 2 17 TOPO TA Cloning Reaction 286
26 2 18 Transformation of Escherichia coli 286
26 3 Example Experiment 286
26 4 Troubleshooting 287
References 288
TJ Identification of Proteins Bound to RNA 291
Emanuele Buratti
27 1 Theoretical Background 291
27 2 Protocol 292
27 2 1 RNA Templates 292
27 2 2 Loading the Beads with RNA 293
27 2 3 Incubation with Protein Mix (Buffer A) 294
27 2 4 Incubation with Protein Mix (Buffer B) 294
27 3 Example Experiment 295
21A Troubleshooting 296
References 296
28 Single-Cell Detection of Splicing Events with Fluorescent
Splicing Reporters 299
Hidehito Kuroyanagi, Akihide Takeuchi, Takayuki Nojima,
and Masatoshi Hagiwara
28 1 Theoretical Background 299
28 1 1 Visualization of Alternative Splicing Patterns with Multiple
Fluorescent Proteins 299
28 1 2 Designing Fluorescent Reporter Minigenes to Monitor Splicing
Patterns 300
28 1 3 Constructing Fluorescent Reporter Minigenes 302
Contents XIII
28 131 MultiSite Gateway System 302
28 132 Other Aspects for Consideration in Minigene Construction 303
28 2 Protocols 303
28 2 1 Constructing Genomic DNA Fragment Cassettes in Entry
Vectors 303
28 211 Primer Design 304
28 212 Performing PCR 304
Protocol 1: Two-Step PCR Amplification of arfB-DNA Fragments 304
'BP' Recombination Reaction and the Selection of Entry Clones 305
Protocol 2: BP Clonase II Reaction and Selection of
Appropriate Entry Clones 305
Modification of Entry Clones (Optional) 305
'LR' Recombination Reaction and Selection of Expression Clones 305
Protocol 3: LR Clonase II Plus Reaction and Selection
of Appropriate Expression Clones 306
Transfection of Cultured Cells and Generation
of Transgenic Animals 306
Checking the Splicing Pattern of the Minigene-Derived mRNAs 306
Protocol 4: RT-PCR Analysis of Minigene-Derived mRNAs 306
28 3 Example Experiments 307
28 4 Troubleshooting 308
References 308
Part Four Analysis of Splicing In Vitro 311
29 The Preparation of HeLa Cell Nuclear Extracts 313
Klaus Hartmuth, Maria A van Santen, Tanja Rbsel,
Berthold Kastner, and Reinhard Luhrmann
29 1 Theoretical Background 313
29 2 Protocols 314
29 3 Example Experiment 318
29 4 Troubleshooting 318
References 318
30 In Vitro Splicing Assays 321
Akila Mayeda and Adrian R Krainer
30 1 Theoretical Background 321
30 2 Protocols 321
30 2 1 Preparation of Pre-mRNA Substrates 321
30 211 Minigene Construction in a Vector for In Vitro Transcription 321
30 212 32P-Labeled Pre-mRNA Preparation by In Vitro Transcription 322
30 2 2 In Vitro Splicing of Pre-mRNAs 323
30 221 Preparation of Reagents 323
30 222 Splicing Reactions 324
30 2 3 Analysis of Splicing Products 325
30 231 Preparation of Reagents 325
30 232 Denaturing PAGE and Autoradiography 326
30 3 Example Experiment 327
30 4 Troubleshooting 327
References 328
31 Assembly and Isolation of Spliceosomal Complexes In Vitro 331
Klaus Hartmuth, Maria A van Santen, Peter Odenwdlder,
and Reinhard Luhrmann
31 1 Theoretical Background 331
31 2 Protocols 332
31 3 Example Experiment 338
XIV Contents
31A Troubleshooting 339
References 340
32 Analysis of Site-Specific RNA-Protein Interactions 343
Nathalie Marmier-Gourrier, Audrey Vautrin, Christiane Branlant,
and Isabelle Behm-Ansmant
32 1 Theoretical Background 343
32 2 Protocols 345
32 2 1 UV Crosslinking and Immunoselection 345
32 211 Chemicals and Enzymes 345
32 212 Buffers 345
32 213 UV Crosslinking 345
32 2 2 RNA Secondary Structure and Footprint Experiments 347
32 221 Probes Used and Properties 347
32 222 Chemicals and Enzymes 348
32 223 Buffers and Reagents 348
32 224 RNA Synthesis 349
32 225 RNA Transcript Renaturation 349
32 226 EMSA 349
32 2 3 Supershift 352
32 231 Chemicals and Enzymes 352
32 232 Buffers 352
32 233 Supershift Experiments 352
32 3 Example Experiments 353
32 4 Troubleshooting 355
References 355
33 Immunoprecipitation and Pull-Down of Nuclear Proteins 359
Natalya Benderska, Chiranthani Sumanasekera, and Stefan Stamm
33 1 Theoretical Background 359
33 1 1 Immunoprecipitation 359
33 1 2 Pull-Down of Proteins 360
33 2 Protocols 360
33 3 Example Experiments 362
33 4 Troubleshooting 363
References 364
34 Analysis of Protein (-RNA) Complexes by (Quantitative) Mass
Spectrometric Analysis 367
Carla Schmidt and Henning Urlaub
34 1 Theoretical Background 367
34 1 1 Mass Spectrometry-Based Identification of Proteins (Proteomics) 367
34 1 2 Sample Preparation and Separation 368
34 121 Additional Separation of Generated Peptides 368
34 122 Separation of Proteins 368
34 1 3 Liquid Chromatography-Coupled Mass Spectrometry (LC-MS) 369
34 1 4 Quantification by Mass Spectrometry 370
34 141 Introduction 370
34 142 Relative Quantification 370
34 143 Absolute Quantification 371
34 1 5 Detection of Post-Translational Modifications by Mass Spectrometry 372
34 151 Introduction 372
34 152 Phosphorylation 372
34 1 6 Detection of Protein-RNA Interactions by Mass Spectrometry 373
34 2 Protocols 373
34 3 Example Experiment 377
34 4 Troubleshooting 377
References 378
Contents XV
Part Five Analysis of Splicing In Vivo 381
35 Fast Cloning of Splicing Reporter Minigenes 383
Zhaiyi Zhang, Amit Khanna, and Stefan Stamm
35 1 Theoretical Background 383
35 1 1 Detection of Alternative Splicing Using Minigenes 383
35 1 2 Site-Specific Recombination 384
35 121 DNA Recombination Sites 384
35 122 Recombination Proteins 385
35 1 3 pSpliceExpress 385
35 2 Protocol 386
35 2 1 Ordering a BAC Clone 386
35 2 2 PCR Primer Design 386
35 221 Forward Primer 386
35 222 Reverse Primer 386
35 223 Nested Primers 386
35 2 3 Performing the PCR 387
35 2 4 Recombination Reaction 387
35 2 5 Transformation of Cells 388
35 2 6 Identification of Positive Clones 388
35 2 7 Transfection of the Minigene 388
35 271 Transfection of HEK293 Cells with Reporter Gene Constructs 389
35 3 Example Experiment 389
35 4 Troubleshooting 390
References 391
36 In Vivo Analysis of Splicing Assays 393
Isabel C Lopez-Mejia andJamal Tazi
36 1 Theoretical Background 393
36 1 1 Studying an Alternative Splicing Event 393
36 1 2 Transfection of Adherent Cell Lines 394
36 2 Protocol 395
36 2 1 Reagents and Solutions 395
36 2 2 Materials and Equipment 395
36 3 Example Experiment 398
36 4 Troubleshooting 398
References 399
37 Coupled Promoter Splicing Systems 401
ManuelJ Muhoz, Manuel de la Mata, and Alberto R Kornblihtt
37 1 Theoretical Background 401
37 2 Protocol 402
37 2 1 Choosing the Promoter of the Minigene: Constitutive
Versus Inducible Promoters 402
37 2 2 The Role of Pol II on Alternative Splicing 403
37 2 3 Transfection of the Alternative Splicing Reporter Minigene 404
37 2 4 RNA Extraction and RT-PCR 405
37 3 Example Experiment 406
37 3 1 Effect of a Slow Pol II Mutant (hC4) in EDI Alternative Splicing 406
17 A Troubleshooting 407
References 407
38 Stable Cell Lines with Splicing Reporters 409
Christian Kroun Damgaard, Soren Lykke-Andersen, andjargen Kjems
38 1 Theoretical Background 409
38 1 1 Plasmid-Based Site-Specific Integration of Splicing Reporters 410
XVI Contents
38 1 2 The Flp Recombinase 410
38 1 3 Induction of Gene Expression 410
38 2 Protocol 411
38 2 1 Cloning 411
38 2 2 Transfection of Recipient Cell Line (HEK293 Flp-In T-REx) 411
38 3 Example Experiment 412
38 3 1 Splicing the Phenotype of Two HIV-1 Minigenes 412
38 4 Troubleshooting 413
References 414
39 Splicing Factor Chi P and ChRI P: Detection of Splicing and Splicing Factors at
Cenes by Chromatin Immunoprecipitation 417
Aparna K Sapra, Fernando Carrillo Oesterreich, Marta Pabis, Imke Listerman,
Nicole Bardehle, and Karla M Neugebauer
39 1 Theoretical Background 417
39 1 1 Cotranscriptional Splicing 417
39 1 2 Chromatin Immunoprecipitation 418
39 1 3 Application of ChIP to Splicing Studies 419
39 1 4 Quantitation of the Immunoprecipitated Nucleic Acids 419
39 2 Protocol 419
39 2 1 Splicing Factor ChIP in S cerevisiae 419
39 211 Buffers and Chemicals Used for ChIP 421
39 2 2 Splicing Factor ChIP in Mammalian Cells 421
39 221 Buffers and Chemicals Used for ChIP 423
39 2 3 ChRIP for Analysis of Cotranscriptional RNA Processing 423
39 231 Additional Buffers and Chemicals Used for ChRIP 424
39 2 4 Data Analysis Using qPCR 424
39 3 Example Experiment 424
39 4 Troubleshooting 426
References 427
40 Yeast Genetics to Investigate the Function of Core Pre-mRNA Splicing
Factors 429
Raymond T O'Keefe and Jean D Beggs
40 1 Theoretical Background 429
40 1 1 The Use of Yeast Saccharomyces cerevisiae as a Model System for Studying
Spliceosomal Factors 429
40 1 2 Yeast Genetics 429
40 1 3 Synthetic Genetic Interactions 430
40 2 Protocol 431
40 2 1 Plasmid Shuffle for Functional Analysis and Production of
Conditional Mutations in Essential Yeast Genes 431
40 211 Construction of Complementing URA3 Plasmid
and a Plasmid for Mutagenesis 431
40 212 Transformation of Diploid Knockout Strain 432
40 213 Yeast Sporulation and Tetrad Dissection 432
40 214 Functional Analysis of Essential Yeast Genes by Plasmid
Shuffle 433
40 2 2 Genetic Interaction Analysis 433
40 221 Construction of Double-Gene URA3 Plasmid 433
40 222 Construction of Double-Knockout Strains 433
40 223 Genetic Screens 434
40 3 Example Experiment 434
40 4 Troubleshooting 435
References 435
Contents XVII
41 Analysis of HIV-1 RNA Splicing 439
Simon Duffy and Alan Cochrane
41 1 Theoretical Background 439
41 1 1 Virus RNA Splicing 439
41 1 2 HIV-1 RNA Splicing 440
41 1 3 Quantitative Reverse Transcription PCR (qRT-PCR) 440
All Protocols 441
41 2 1 qRT-PCR Primer Design 441
41 211 Primers for Analysis of HIV-1 Transcripts 441
41 2 2 Transfection of Virus/Provirus Vector DNA into Target Cells 441
41 2 3 Secreted Alkaline Phosphatase (SEAP) Assay 442
All A RNA Isolation and cDNA Synthesis 442
41 2 5 qRT-PCR Reaction Conditions and Analysis 443
41 2 6 Analysis of Splice Site Utilization 444
41 3 Example Experiment 446
41 4 Troubleshooting 447
References 448
42 In Vivo Analysis of Plant Intron Splicing 451
Craig G Simpson, Michele Liney, Diane Davidson, Dominika Lewandowska,
Maria Kalyna, Sean Chapman, Andrea Barta, and John W S Brown
42 1 Theoretical Background 451
42 1 1 Plant Splicing Analysis In Vivo 451
42 1 2 Splicing of Plant and Animal Introns in Reciprocal Systems 451
42 1 3 Plant Splicing Reporter Constructs 452
42 1 4 Expression of trans-Acting Factors 453
All Protocols 453
42 2 1 Transfection of Plasmid DNA into Plant Protoplasts 453
42 2 2 Analyzing the Results of In vivo Splicing Analysis 459
42 221 RNA Extraction 459
Mill RT-PCR Analysis 459
42 223 Western Analysis 460
42 224 Visualizing GFP Expression 460
42 3 Example Experiment 461
42 4 Troubleshooting 461
References 462
43 Modification State-Specific Antibodies 465
Jordan B Fishman, Olga Kelemen, and Eric A Berg
43 1 Theoretical Background 465
43 2 Protocol 466
43 2 1 Selection of the Target Site and Peptide Immunogen Design 466
43 2 2 Peptide Synthesis 467
43 2 3 Peptide Immunogen-Carrier Protein Conjugation 469
43 2 4 Immunization, Antibody Purification, and Immunodepletion 469
43 2 5 The Human SMAD2 Protein C-Terminal Domain 470
43 3 Example Experiment 471
43 4 Troubleshooting 472
References 473
44 Analysis of Alternative Splicing in Drosophila Genetic Mosaics 475
Shihuang Su, Diana O'Day, Shanzhi Wang, and William Mattox
44 1 Theoretical Background 475
44 1 1 Reporter Genes for Splicing in Living Organisms 475
44 1 2 The Introduction of Splicing Reporters into the Drosophila Genome 475
44 1 3 The Application of Genetic Mosaics to the Analysis of Alternative
Pre-mRNA Splicing 476
XVIII Contents
44 2 Protocol 476
44 2 1 Transgenes Used in Flip-Out Studies 476
44 2 2 Generation of Mosaic Flies 478
44 2 3 Immunostaining of Drosophila Tissues 478
44 3 Example Experiment 479
44 4 Troubleshooting 479
References 479
Part Six Manipulation of Splicing Events' 481
45 Antisense Derivatives of U7 Small Nuclear RNA as Modulators
of Pre-mRNA Splicing 483
Kathrin Meyer and Daniel Sch'umperli
45 1 Theoretical Background 483
45 1 1 What Makes U7 snRNA a Suita ble In Vivo Splicing Modulation Tool? 483
45 1 2 Strategic Considerations 485
45 1 3 Gene Transfer and Regulated Expression 487
45 2 Protocols 488
45 3 Example Experiment 492
45 3 1 Mutagenic PCR (Protocol 1) with Four Different Primers 492
45 4 Troubleshooting 492
References 493
46 Screening for Alternative Splicing Modulators 497
Peter Stoilov
46 1 Theoretical Background 497
46 1 1 Overview of In Vivo Splicing Reporters 497
46 1 2 Two-Color Fluorescent Reporters for Splicing and Translation 498
46 1 3 Data Acquisition and Processing 409
46 1 4 Hit Validation 500
46 2 Protocols 500
46 2 1 Equipment and Materials 501
46 211 Equipment 501
46 212 Materials 501
46 2 2 Compound Plate Layout 50?
46 2 3 Cell Plating and Compound Addition 502
46 2 4 Imaging 502
46 2 5 Image Processing and Data Analysis 502
46 2 6 RT-PCR Validation 503
46 261 RNA Preparation 503
46 262 First-Strand cDNA Synthesis 503
46 263 PCR Amplification 504
46 264 Capillary Electrophoresis 504
46 3 Example Experiment 504
46 4 Troubleshooting 505
References 507
47 Use of Oligonucleotides to Change Splicing 511
Annemieke Aartsma-Rus and Judith C T van Deutekom
47 1 Theoretical Background 511
47 1 1 Antisense-Mediated Modulation of Splicing 511
47 1 2 AON Design 513
47 1 3 AON Chemistry 514
47 1 4 Transfection in Cultured Cells 514
47 1 5 RNA Analysis 515
All Protocol 515
47 2 1 AONs 515
Contents XIX
47 2 2 Transfection 516
47 2 3 RNA Isolation 517
47 2 4 RT-PCR Analysis 517
47 2 5 Determining Exon Skipping Efficiency 518
47 3 Example Experiment 518
47 4 Troubleshooting 518
References 519
48 Changing Signals to the Spliceosome 523
Denise R Cooper and Niketa A Patel
48 1 Theoretical Background 523
48 1 1 Identification of Extracellular Stimulated Signaling Pathways
Altering Spliceosomal Proteins 523
48 1 2 Identification of tis-Elements and trans-Factors Targeted by
Kinase Pathways 525
48 1 3 To Establish Whether a Kinase Regulating an Alternative
Exon is Activated by a Hormone 525
48 1 4 Use of siRNA to Establish the Role of a Kinase and Spliceosomal Protein
in a Splicing Event 526
48 1 5 Use of 2'-O-Methoxyethyl (MOE) Antisense Oligonucleotides to Identify
Relevant cis-Elements in the Sequence 526
48 1 6 Identifying the Minimum Boundaries of the Genomic Sequence Regulated
by Stimuli for Cloning into a Heterologous Minigene 528
48 1 7 Physiological Readout of Splice Variant Function 528
48 1 8 Prediction of Splicing Relevance Using Knockout Tissue
or Tissue from Mutant or Disease States 528
48 2 Protocols 528
48 2 1 Cell Culture 528
48 2 2 Transfections 529
48 2 3 Western Blot Analysis 529
ASIA Immunoprecipitation 530
48 241 Buffer solutions 530
48 2 5 Minigenes 530
48 2 6 RT-PCR Analysis 531
48 2 7 Inhibitors 532
48 3 Example Experiment 532
48 4 Troubleshooting 533
References 533
Part Seven Bioinformatic Analysis of Splicing
49 Overview of Splicing Relevant Databases 537
Pierre de la Grange
49 1 Theoretical Background 537
49 1 1 Alternative Splicing Databases: Interest 537
49 1 2 Alternative Splicing Databases: Common Strategy 537
49 1 3 Description of Alternative Splicing Databases 538
49 1 4 The UCSC Genome Browser 538
49 2 Protocol 538
49 3 Example Experiment 541
49 4 Troubleshooting 541
References 542
50 Analysis of RNA Transcripts by High-Throughput RNA Sequencing 545
Paolo Ribeca, Vincent Lacroix, Michael Sammeth, and Roderic Guigb
50 1 Theoretical Background 545
50 1 1 Reads, Mapping, and Mappability 545
XX Contents
50 1 2 Quantification of AS Events and Read Deconvolution 546
50 2 Protocol 548
50 2 1 Experimental Protocol 548
50 2 2 Pre-Processing of the Reads 548
50 2 3 Mapping of the Reads 550
50 231 Reference Indexing 550
50 2 4 Quantification of Abundances 551
50 3 Example Experiment 552
50 4 Troubleshooting 553
References 554
51 Identification of Splicing Factor Target Genes by
High-Throughput Sequencing 557
Chaolin Zhang and Michael Q Zhang
51 1 Theoretical Background 557
51 1 1 Transcriptome Analysis by mRNA-Seq 557
51 1 2 Available NGS Platforms 558
51 1 3 Short Reads Mapping 558
51 1 4 Single-End versus Paired-End mRNA-Seq 560
51 2 Protocol 560
51 2 1 mRNA-Seq Experiment Design 560
51 2 2 Sequence Alignment 561
51 2 3 Read Counting and Statistical Analysis 561
51 2 4 Visualization of Data 562
51 3 Example Experiment 562
51A Troubleshooting 563
References 563
52 Bioinformatic Analysis of Splicing Events 567
Zhaiyi Zhang and Stefan Stamm
52 1 Theoretical Background 567
52 1 1 General Databases 567
52 1 2 Splice Site Analysis 568
52 1 3 Exonic and Intronic Splicing Elements 568
52 1 4 Secondary Structure 570
52 2 Protocol 570
52 2 1 Detecting a New Sequence Using BLAT 570
52 2 2 Determine Regulatory Elements in RNA Sequence 570
52 3 Example Experiment 571
52 4 Troubleshooting 572
References 573
53 Analysis of Pre-mRNA Secondary Structures and Alternative
Splicing 575
Michael Hiller
53 1 Theoretical Background 575
53 1 1 Alternative Splicing and Secondary Structures 575
53 1 2 Computational Prediction of Secondary Structures 576
53 2 Protocol 576
53 2 1 Input Sequences 576
53 2 2 Predicting Single-Stranded Regions 577
53 221 Computing Base-Pair Probability Plots 577
53 222 Computing PU Values 577
53 2 3 Predicting if a Mutation Leads to Structural Changes 578
53 231 Comparing Base-Pair Probability Plots 578
53 232 Computing PU Values 579
Contents XXI
53 2 4 Finding Alternative Splice Events that Overlap Evolutionarily
Conserved Secondary Structures 579
53 3 Example Experiment 579
53 4 Troubleshooting 580
References 580
54 Structure Prediction for Alternatively Spliced Proteins 583
Lukasz Kozlowski, Jerzy Orlowski, andJanusz M Bujnicki |
| any_adam_object | 1 |
| author2 | Stamm, Stefan |
| author2_role | edt |
| author2_variant | s s ss |
| author_facet | Stamm, Stefan |
| building | Verbundindex |
| bvnumber | BV036860605 |
| classification_rvk | WC 4460 |
| ctrlnum | (OCoLC)699751343 (DE-599)DNB1006150145 |
| dewey-full | 611.01816 572.8845 |
| dewey-hundreds | 600 - Technology (Applied sciences) 500 - Natural sciences and mathematics |
| dewey-ones | 611 - Human anatomy, cytology, histology 572 - Biochemistry |
| dewey-raw | 611.01816 572.8845 |
| dewey-search | 611.01816 572.8845 |
| dewey-sort | 3611.01816 |
| dewey-tens | 610 - Medicine and health 570 - Biology |
| discipline | Biologie Medizin |
| format | Book |
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| id | DE-604.BV036860605 |
| illustrated | Illustrated |
| indexdate | 2024-07-20T10:53:42Z |
| institution | BVB |
| isbn | 9783527326068 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-020776373 |
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| physical | XXXVII, 622 S. graph. Darst. |
| publishDate | 2012 |
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| publisher | Wiley-VCH |
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| spelling | Alternative pre-mRNA splicing theory and protocols ed. by Stefan Stamm ... RNA splicing Weinheim Wiley-VCH 2012 XXXVII, 622 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Auch angekündigt als: RNA splicing RNS-Spleißen (DE-588)4201877-8 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content RNS-Spleißen (DE-588)4201877-8 s DE-604 Stamm, Stefan edt X:MVB text/html http://deposit.dnb.de/cgi-bin/dokserv?id=3527478&prov=M&dok_var=1&dok_ext=htm Inhaltstext HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020776373&sequence=000004&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Alternative pre-mRNA splicing theory and protocols RNS-Spleißen (DE-588)4201877-8 gnd |
| subject_GND | (DE-588)4201877-8 (DE-588)4143413-4 |
| title | Alternative pre-mRNA splicing theory and protocols |
| title_alt | RNA splicing |
| title_auth | Alternative pre-mRNA splicing theory and protocols |
| title_exact_search | Alternative pre-mRNA splicing theory and protocols |
| title_full | Alternative pre-mRNA splicing theory and protocols ed. by Stefan Stamm ... |
| title_fullStr | Alternative pre-mRNA splicing theory and protocols ed. by Stefan Stamm ... |
| title_full_unstemmed | Alternative pre-mRNA splicing theory and protocols ed. by Stefan Stamm ... |
| title_short | Alternative pre-mRNA splicing |
| title_sort | alternative pre mrna splicing theory and protocols |
| title_sub | theory and protocols |
| topic | RNS-Spleißen (DE-588)4201877-8 gnd |
| topic_facet | RNS-Spleißen Aufsatzsammlung |
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