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: www.wiley-vch.de/home/splicing
Companion Website: www.wiley-vch.de/home/splicing
INTRODUCTION
Splicing in the RNA World (Emanuele Buratti, Maurizio Romano and Francisco
E. Baralle)
RNPs, small RNAs, miRNAs (Michaela Beitzinger and Gunter Meister)
RNA Elements Involved in Splicing (William F. Mueller and Klemens J.
Hertel)
A Structural Biology Perspective of Proteins Involved in Splicing Regulation
(Antoine Cléry and Frederic Allain)
The Spliceosome in Constitutive Splicing (Patrizia Fabrizio and Reinhard
Lührmann)
The Use of Saccharomyces cerevisiae to Study the Mechanism of pre-mRNA
Splicing (Brian C. Rymond)
Challenges in Plant Alternative Splicing (Andrea Barta, Yamile Marquez and
John WS Brown)
Alternative Splice Site Selection (Miguel B. Coelho and Christopher WJ
Smith)
Integration of Splicing with Nuclear and Cellular Events (Aparna K. Sapra
and Carla Neugebauer)
Splicing and Disease (Emanuele Buratti and Francisco E. Baralle)
From Bedside to Bench: How to Analyze a Splicing Mutation (Marco Baralle and
Diana Baralle)
Analysis of Splicing Problems (Stefan Stamm)
BASIC METHODS
Ultracentrifugation in the Analysis and Purification by of Spliceosomes
Assembled in Vitro
(Klaus Hartmuth, Maria A. van Santen and Reinhard Lührmann)
Chemical Synthesis of RNA (Claudia Höbartner)
RNA Interference (siRNA, ShRNA) (Daphne S. Cabianca and Davide Gabellini)
Expression and Purification of Splicing Proteins (James Stévenin and Cyril
F. Bourgeois)
Detection of RNA-Protein Complexes By Electrophoretic Mobility Shift Assay
(Manli Shen and Michael G. Fried)
Functional Analysis of large Exonic Sequences through Iterative in Vivo
Selection (Natalia N. Singh, Joonbae Seo and Ravindra N. Singh)
Identification of Splicing cis-Elements through an Ultra-Refined Antisense
Microwalk (Ravindra N. Singh and Natalia N. Singh)
Genomic SELEX to Identify RNA Targets of Plant RNA Binding Proteins (Olga
Bannikova and Andrea Barta)
DETECTION OF SPLICING EVENTS
Quantification of Alternative Splice Variants (Miriam LLorian and
Christopher WJ Smith)
High-Throughput Analysis of Alternative Splicing by RT-PCR (Roscoe Klinck,
Benoit Chabot and Sherif Abou Elela)
Monitoring Changes in Plant Alternative Splicing Events (Craig G. Simpson,
Naeem Hasan Syed, Sujatha Manthri, John D. Fuller, Monika Maronova, Branislav
Kusenda, Maria Kalyna, Andrea Barta and John WS Brown)
Array Analysis (Pierre de la Grange)
The CLIP Method to Study Protein-RNA Interactions in Intact Cells and
Tissues (James Tollervey and Jernej Ule)
RNA: Protein Crosslinking and Immunoprecipitation in Yeast (CLIP) (Branislav
Kusenda and Andrea Barta)
Identification of Proteins Bound to RNA (Emanuele Buratti)
Single Cell Detection of Splicing Events with Fluorescent Splicing Reporters
(Hidehito Kuroyanagi, Akihide Takeuchi, Takayuki Nojima and Masatoshi
Hagiwara)
ANALYSIS OF SPLICING IN VITRO
The Preparation of HeLa Cell Nuclear Extracts (Klaus Hartmuth, Maria A. van
Santen, Tanja Rösel, Berthold Kastner and Reinhard Lührmann)
In Vitro Splicing Assays (Akila Mayeda and Adrian R. Krainer)
Assembly of Spliceosomes in Vitro(Klaus Hartmuth, Maria A. van Santen, Peter
Odenwälder and Reinhard Lührmann)
Analysis of Site-Specific RNA-Protein Interactions (Nathalie
Marmier-Gourrier, Audrey Vautrin, Christiane Branlant and Isabelle
Behm-Ansmant)
Immunoprecipitation and Pull-Down of Nuclear Proteins
(Natalya Benderska, Chiranthani Sumanasekera and Stefan Stamm)
Analysis of Protein (RNA) Complexes by (Quantitative) Mass Spec Analysis
(Carla Schmidt and Henning Urlaub)
ANALYSIS OF SPLICING IN VIVO
Fast cloning of splicing reporter minigenes (Zhaiyi Zhang, Amit Khanna and
Stefan Stamm)
In Vivo Splicing Assays (Isabel Cristina López Mejía and Jamal Tazi)
Coupled Promoter Splicing Systems (Manuel J. Muñoz, Manuel de la Mata and
Alberto R. Kornblihtt)
Stable Cell Lines With Splicing Reporters (Christian Kroun Damgaard, Søren
Lykke-Andersen and Jørgen Kjems)
Splicing Factor ChIP and ChRIP: Detection of Splicing and Splicing Factors
at Genes by Chromatin Immunoprecipitation (Aparna K. Sapra, Fernando Carrillo
Oesterreich, Marta Pabis, Imke Listerman, Nicole Bardehle and Karla M.
Neugebauer)
Yeast Genetics to Investigate the Function of Core Pre-mRNA Splicing Factors
(Raymond T. O?Keefe and Jean D. Beggs)
Analysis of HIV-1 RNA Splicing (Simon Duffy and Alan Cochrane)
In Vivo Analysis of Plant Intron Splicing (Craig G. Simpson, Michele Liney,
Diane Davidson, Dominika Lewandowska, Maria Kalyna, Sean Chapman, Andrea
Barta and John WS Brown)
Modificaton State-Specific Antibodies (Jordan B. Fishman, Olga Kelemen and
Eric A. Berg)
Analysis of Alternative Splicing in Drosophila Genetic Mosaics (Shihuang Su,
Diana O?Day, Shanzhi Wang and William Mattox)
MANIPULATION OF SPLICING EVENTS
Antisense Derivatives of U7 Small Nuclear RNA as Modulators of Pre-mRNA
Splicing (Kathrin Meyer and Daniel Schümperli)
Screening for Alternative Splicing Modulators (Peter Stoilov)
Use of Oligonucleotides to Change Splicing (Annemieke Aartsma-Rus and Judith
C.T. van Deutekom)
Changing Signals to the Spliceosome (Denise R. Cooper and Niketa A. Patel)
BIOINFORMATIC ANALYSIS OF SPLICING
Overview of Splicing Relevant Databases (Pierre de la Grange)
High Throughput Sequencing (Paolo Ribeca, Vincent Lacroix, Michael Sammeth
and Roderic Guigó)
Identification of Splicing Factor Target Genes by High Throughput
Sequencing
(Chaolin Zhang and Michael Q. Zhang)
Bioinformatic Analysis of Splicing Events (Zhaiyi Zhang and Stefan Stamm)
Analysis of Pre-mRNA Secondary Structures and Alternative Splicing (Michael
Hiller)
Structure Prediction for Alternatively Spliced Proteins
(Lukasz Kozlowski, Jerzy Orlowski and Janusz M. Bujnicki)
Comparative Genomics Methods for the Prediction of Small RNA Binding Sites
(Rym Kachouri-Lafond and Mihaela Zavolan)
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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