Spliceosomal Pre-mRNA Splicing

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.

The Pre-mRNA Splicing Reaction.- Diversity and Evolution of Spliceosomal Systems.- Mechanisms of Spliceosomal Assembly.- Alternative Pre-mRNA Splicing.- Regulation of Alternative Pre-mRNA Splicing.- Introduction to Co-Transcriptional RNA Splicing.- Chromatin and Splicing.- Preparation of Splicing Competent Nuclear Extracts.- Preparation of Yeast Whole Cell Splicing Extract.- Efficient Splinted Ligation of Synthetic RNA Using RNA Ligase.- In Vitro Assay of Pre-mRNA Splicing in Mammalian Nuclear Extract.- Kinetic Analysis of In Vitro Pre-mRNA Splicing in HeLa Nuclear Extract.- In Vitro Systems for Coupling RNAP II Transcription to Splicing and Polyadenylation.- Isolation and Accumulation of Spliceosomal Assembly Intermediates.- Complementation of U4 snRNA in S. cerevisiae Splicing Extracts for Biochemical Studies of snRNP Assembly and Function.- Expression and Purification of Splicing Proteins from Mammalian Cells.- Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis.- Cell-Based Splicing of Minigenes.- Quantifying the Ratio of Spliceosome Components Assembled on Pre-mRNA.- Antisense Methods to Modulate Pre-mRNA Splicing.- Using Yeast Genetics to Study Splicing Mechanisms.- Medium Throughput Analysis of Alternative Splicing by Fluorescently Labeled RT-PCR.- Chromatin Immunoprecipitation Approaches to Determine Co-Transcriptional Nature of Splicing.- Computational Approaches to Mine Publicly Available Databases.- Approaches to Link RNA Secondary Structures with Splicing Regulation.- Methods to Study Splicing from High-Throughput RNA Sequencing Data.- Global Protein-RNA Interaction Mapping at Single Nucleotide Resolution by iCLIP-Seq.- Predicting Alternative Splicing.