Description
Providing a guide to classical experimental approaches to decipher splicing mechanisms and experimental strategies that rely on novel multi-disciplinary approaches, Spliceosomal Pre-mRNA Splicing: Methods and Protocols describes the theory of alternative pre-mRNA splicing in seven introductory chapters and then introduces protocols and their theoretical background relevant for a variety of experimental research. These protocol chapters cover basic methods to detect splicing events, analyses of alternative pre-mRNA splicing in vitro and in vivo manipulation of splicing events and high-throughput and bioinformatic analyses of alternative splicing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, Spliceosomal Pre-mRNA Splicing: Methods and Protocols will aid newcomers and seasoned molecular biologists in understanding the fascinating world of alternative splicing with the ultimate goal of paving the way for many new discoveries to come. Part I: Introductory Chapters 1. The Pre-mRNA Splicing Reaction Somsakul Wongpalee and Shalini Sharma 2. Diversity and Evolution of Spliceosomal Systems Scott William Roy and Manuel Irimia 3. Mechanisms of Spliceosomal Assembly Ni-ting Chiou and Kristen W. Lynch 4. Alternative Pre-mRNA Splicing Stacey D. Wagner and J. Andrew Berglund 5. Regulation of Alternative Pre-mRNA Splicing Miguel B. Coelho and Christopher W.J. Smith 6. Introduction to Co-Transcriptional RNA Splicing Evan C. Merkhofer, Peter Hu, and Tracy L. Johnson 7. Chromatin and Splicing Nazmul Haque and Shalini Oberdoerffer Part II: Methods Chapters 8. Preparation of Splicing Competent Nuclear Extracts Chiu-Ho T. Webb and Klemens J. Hertel 9. Preparation of Yeast Whole Cell Splicing Extract Elizabeth A. Dunn and Stephen D. Rader 10. Efficient Splinted Ligation of Synthetic RNA Using RNA Ligase Martha R. Stark and Stephen D. Rader 11. In Vitro Assay of Pre-mRNA Splicing in Mammalian Nuclear Extract Maliheh Movassat, William F. Mueller, and Klemens J. Hertel 12. Kinetic Analysis of In Vitro Pre-mRNA Splicing in HeLa Nuclear Extract William F. Mueller and Klemens J. Hertel 13. In Vitro Systems for Coupling RNAP II Transcription to Splicing and Polyadenylation Eric G. Folco and Robin Reed 14. Isolation and Accumulation of Spliceosomal Assembly Intermediates Janine O. Ilagan and Melissa S. Jurica 15. Complementation of U4 snRNA in S. cerevisiae Splicing Extracts for Biochemical Studies of snRNP Assembly and Function Martha R. Stark and Stephen D. Rader 16. Expression and Purification of Splicing Proteins from Mammalian Cells Eric Allemand and Michelle L. Hastings 17. Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis Eric G. Anderson and Aaron A. Hoskins 18. Cell-Based Splicing of Minigenes Sarah A. Smith and Kristen W. Lynch 19. Quantifying the Ratio of Spliceosome Components Assembled on Pre-mRNA Noa Neufeld, Yehuda Brody, and Yaron Shav-Tal 20. Antisense Methods to Modulate Pre-mRNA Splicing Joonbae Seo, Eric W. Ottesen, and Ravindra N. Singh 21. Using Yeast Genetics to Study Splicing Mechanisms Munshi Azad Hossain and Tracy L. Johnson 22. Medium Throughput Analysis of Alternative Splicing by Fluorescently Labeled RT-PCR Ryan Percifield, Daniel Murphy, and Peter Stoilov 23. Chromatin Immunoprecipitation Approaches to Determine Co-Transcriptional Nature of Splicing Nicole I. Bieberstein, Korinna Straube, and Karla M. Neugebauer 24. Computational Approaches to Mine Publicly Available Databases Rodger B. Voelker, William A. Cresko, and J. Andrew Berglund 25. Approaches to Link RNA Secondary Structures with Splicing Regulation Mireya Plass and Eduardo Eyras 26. Methods to Study Splicing from High-Throughput RNA Sequencing Data Gael P. Alamancos, Eneritz Agirre, and Eduardo Eyras 27. Global Protein-RNA Interaction Mapping at Single Nucleotide Resolution by iCLIP-Seq Chengguo Yao, Lingjie Weng, and Yongsheng Shi 28. Predicting Alternative Splicing Yoseph Barash and Jorge Vaquero Garcia
