Alternative pre-mRNA splicing: theory and protocols

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 beenlinked 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. INDICE: INTRODUCTION Splicing in the RNA World (Emanuele Buratti, MaurizioRomano 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 Clery and Frederic Allain) The Spliceosome in Constitutive Splicing (Patrizia Fabrizio and Reinhard Luhrmann) 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 Luhrmann) Chemical Synthesis of RNA (Claudia Hobartner) RNA Interference (siRNA, ShRNA) (Daphne S. Cabianca and Davide Gabellini) Expression and Purification of Splicing Proteins (James Stevenin 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 Immunoprecipitationin 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 Rosel, Berthold Kastner and Reinhard Luhrmann) In Vitro Splicing Assays (AkilaMayeda and Adrian R. Krainer) Assembly of Sp

• ISBN: 978-3-527-32606-8
• Editorial: Wiley-VCH
• Encuadernacion: Rústica
• Páginas: 660
• Fecha Publicación: 07/12/2011
• Nº Volúmenes: 1
• Idioma: Inglés