Overview

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 ...
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Alternative pre-mRNA Splicing: Theory and Protocols

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Overview

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: wiley-vch.de/home/splicing
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Editorial Reviews

From the Publisher
"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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Product Details

  • ISBN-13: 9783527647989
  • Publisher: Wiley
  • Publication date: 8/1/2012
  • Sold by: Barnes & Noble
  • Format: eBook
  • Edition number: 1
  • Pages: 660
  • File size: 10 MB

Meet 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 Lührmann is Director at the Max Planck Institute for Biophysical Chemistry in Göttingen where he is head of the Department of Cellular Biochemistry. He is an Honorary Professor at the University of Göttingen. He has studied chemistry at the University of Münster 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.
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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-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

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