All eukaryotic messenger RNAs are derived from precursors, the pre-mRNAs, by an extensive series of nuclear modifi cations. Most notably, the removal of introns and the joining of the remaining exons in the process of pre-mRNA splicing is essential to generate mRNAs that are suitable templates for translation. The vast majority of human pre-mRNAs undergo alternative splicing, in which different RNA parts are either included or omitted in the mature mRNA. This process allows individual genes to generate multiple proteins and makes alternative pre-mRNA splicing a central element in gene regulation. Although pre-mRNA splicing occurs with high fi delity, an increasing number of diseases have been linked to defects that lead to mis-spliced mRNAs.
This book was written for graduate and medical students, as well as clinicians and postdoctoral researchers. It describes the theory of alternative pre-mRNA splicing in twelve introductory chapters and then introduces protocols and their theoretical background relevant for experimental research. These 43 practical chapters cover: Basic methods, Detection of splicing events, Analysis of alternative pre-mRNA splicing in vitro and in vivo, Manipulation of splicing events, and Bioinformatic analysis of alternative splicing.
A theoretical introduction and practical guide for molecular biologists, geneticists,clinicians and every researcher interested in alternative splicing.
TOC:
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
2 RNPs, Small RNAs, and miRNAs 13
Michaela Beitzinger and Gunter Meister
3 RNA Elements Involved in Splicing 21
William F. Mueller and Klemens J. Hertel
4 A Structural Biology Perspective of Proteins Involved in Splicing Regulation 33
Antoine Clery and Frederic H.-T. Allain
5 The Spliceosome in Constitutive Splicing 49
Patrizia Fabrizio and Reinhard L€uhrmann
6 The Use of Saccharomyces cerevisiae to Study the Mechanism of pre-mRNA Splicing 65
Brian C. Rymond
7 Challenges in Plant Alternative Splicing 79
Andrea Barta, Yamile Marquez, and John W.S. Brown
8 Alternative Splice Site Selection 93
Miguel B. Coelho and Christopher W.J. Smith
9 Integration of Splicing with Nuclear and Cellular Events 109
Aparna K. Sapra and Karla Neugebauer
10 Splicing and Disease 119
Emanuele Buratti and Francisco E. Baralle
11 From Bedside to Bench: How to Analyze a Splicing Mutation 129
Marco Baralle and Diana Baralle
Part Two Basic Methods 139
12 Analysis of Common Splicing Problems 141
Stefan Stamm
13 Ultracentrifugation in the Analysis and Purification of Spliceosomes Assembled In Vitro 147
Klaus Hartmuth, Maria A. van Santen, and Reinhard L€uhrmann
14 Chemical Synthesis of RNA 155
Claudia H€obartner
15 RNA Interference (siRNA, shRNA) 165
Daphne S. Cabianca and Davide Gabellini
16 Expression and Purification of Splicing Proteins 175
James Stevenin and Cyril F. Bourgeois
17 Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay 183
Manli Shen and Michael G. Fried
18 Functional Analysis of Large Exonic Sequences Through Iterative In Vivo Selection 201
Ravindra N. Singh and Natalia N. Singh
19 Identification of Splicing cis-Elements Through an Ultra-Refined Antisense Microwalk 211
Natalia N. Singh, Joonbae Seo, and Ravindra N. Singh
20 Genomic SELEX to Identify RNA Targets of Plant RNA-Binding Proteins 219
Olga Bannikova, Maria Kalyna, and Andrea Barta
Part Three Detection of Splicing Events 227
21 Quantification of Alternative Splice Variants 229
Miriam Llorian and Christopher W.J. Smith
22 High-Throughput Analysis of Alternative Splicing by RT-PCR 239
Roscoe Klinck, Benoit Chabot, and Sherif Abou Elela
23 Monitoring Changes in Plant Alternative Splicing Events 249
Craig G. Simpson, Naeem Hasan Syed, Sujatha Manthri, John D. Fuller, Monika Maronova, Branislav Kusenda, Maria Kalyna, Andrea Barta, and John W.S. Brown
24 Array Analysis 261
Pierre de la Grange
25 The CLIP Method to Study Protein–RNA Interactions in Intact Cells and Tissues 269
James Tollervey and Jernej Ule
26 RNA–Protein Crosslinking and Immunoprecipitation (CLIP) in Schizosaccharomyces pombe 281
Branislav Kusenda and Andrea Barta
27 Identification of Proteins Bound to RNA 291
Emanuele Buratti
28 Single-Cell Detection of Splicing Events with Fluorescent Splicing Reporters 299
Hidehito Kuroyanagi, Akihide Takeuchi, Takayuki Nojima, and Masatoshi Hagiwara
Part Four Analysis of Splicing In Vitro 311
29 The Preparation of HeLa Cell Nuclear Extracts 313
Klaus Hartmuth, Maria A. van Santen, Tanja R€osel, Berthold Kastner, and Reinhard L€uhrmann
30 In Vitro Splicing Assays 321
Akila Mayeda and Adrian R. Krainer
31 Assembly and Isolation of Spliceosomal Complexes In Vitro 331
Klaus Hartmuth, Maria A. van Santen, Peter Odenw€alder, and Reinhard L€uhrmann
32 Analysis of Site-Specific RNA–Protein Interactions 343
Nathalie Marmier-Gourrier, Audrey Vautrin, Christiane Branlant, and Isabelle Behm-Ansmant
33 Immunoprecipitation and Pull-Down of Nuclear Proteins 359
Natalya Benderska, Chiranthani Sumanasekera, and Stefan Stamm
34 Analysis of Protein (-RNA) Complexes by (Quantitative) Mass Spectrometric Analysis 367
Carla Schmidt and Henning Urlaub
Part Five Analysis of Splicing In Vivo 381
35 Fast Cloning of Splicing Reporter Minigenes 383
Zhaiyi Zhang, Amit Khanna, and Stefan Stamm
36 In Vivo Analysis of Splicing Assays 393
Isabel C. Lopez-Mejía and Jamal Tazi
37 Coupled Promoter Splicing Systems 401
Manuel J. Mu~noz, Manuel de la Mata, and Alberto R. Kornblihtt
38 Stable Cell Lines with Splicing Reporters 409
Christian Kroun Damgaard, Søren Lykke-Andersen, and Jørgen Kjems
39 Splicing Factor ChIP and ChRIP: Detection of Splicing and Splicing Factors at Genes by Chromatin Immunoprecipitation 417
Aparna K. Sapra, Fernando Carrillo Oesterreich, Marta Pabis, Imke Listerman, Nicole Bardehle, and Karla M. Neugebauer
40 Yeast Genetics to Investigate the Function of Core Pre-mRNA Splicing Factors 429
Raymond T. O.Keefe and Jean D. Beggs
41 Analysis of HIV-1 RNA Splicing 439
Simon Duffy and Alan Cochrane
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
43 Modification State-Specific Antibodies 465
Jordan B. Fishman, Olga Kelemen, and Eric A. Berg
44 Analysis of Alternative Splicing in Drosophila Genetic Mosaics 475
Shihuang Su, Diana O.Day, Shanzhi Wang, and William Mattox
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
46 Screening for Alternative Splicing Modulators 497
Peter Stoilov
47 Use of Oligonucleotides to Change Splicing 511
Annemieke Aartsma-Rus and Judith C.T. van Deutekom
48 Changing Signals to the Spliceosome 523
Denise R. Cooper and Niketa A. Patel
50 Analysis of RNA Transcripts by High-Throughput RNA Sequencing 545
Paolo Ribeca, Vincent Lacroix, Michael Sammeth, and Roderic Guigo
51 Identification of Splicing Factor Target Genes by High-Throughput Sequencing 557
Chaolin Zhang and Michael Q. Zhang
52 Bioinformatic Analysis of Splicing Events 567
Zhaiyi Zhang and Stefan Stamm
53 Analysis of Pre-mRNA Secondary Structures and Alternative Splicing 575
Michael Hiller
54 Structure Prediction for Alternatively Spliced Proteins 583
Lukasz Kozlowski, Jerzy Orlowski, and Janusz M. Bujnicki
55 Comparative Genomics Methods for the Prediction of Small RNA-Binding Sites 593
Rym Kachouri-Lafond and Mihaela Zavolan
References 600
Appendices 603
Appendix A1: Yeast Nomenclature Systematic Open Reading Frame (ORF) Designations 605
Min Chen and Brian Rymond
A1.1 Protein-Coding Genes 605
A1.2 Recombinant Derivatives 606
A1.3 Proteins 606
A1.4 Noncoding Genes, Genes Not Encoded by Nuclear Chromosomal DNA, and other Chromosomal Features 606
A1.5 Yeast Strains 607
References 607
Appendix A2: Glossary 609
Index 615