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Alternative Pre-mRNA Splicing

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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. Website:
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Table of 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 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-Mejia 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, Soren Lykke-Andersen, and Jorgen 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

About the Author

Stefan Stamm is Associate Professor in the Department for Molecular and Cellular Biochemistry at the University of Kentucky. He studied Biochemistry in Hannover (Germany) and did the practical work for his PhD as well as postdoctoral work at the Cold Spring Harbor Laboratory, NY. His research focuses on mechanisms and regulation of alternative splicing, with the aim to apply the fi ndings to the Prader-Willi Syndrome and Spinal Muscular Atrophy. Chris Smith is Professor of RNA Molecular Biology in the Department of Biochemistry at the University of Cambridge. His PhD research in Biochemistry was carried out at the University of London, followed by postdoctoral research at Harvard Medical School. His research interests encompass the mechanisms, regulation and function of alternative splicing. Reinhard Luhrmann is Director at the Max Planck Institute for Biophysical Chemistry in Gottingen where he is head of the Department of Cellular Biochemistry. He is an Honorary Professor at the University of Gottingen. He has studied chemistry at the University of Munster where he also received his PhD. He worked as a postdoctoral fellow at the Max Planck Institute for Molecular Genetics in Berlin. His research focuses on the structure and mechanism of the spliceosome.


"This book, prepared within the context of the EURASNET framework, is the first of its kind and, as such, an important piece of scientific literature, which will inspire more investigators to enter the rising field of alternative pre-mRNA splicing." ( Biotechnology Journal, April 2012)

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