Spliceosomal Pre-mRNA Splicing Methods and Protocols

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