mRNA Processing and Metabolism Methods and Protocols

About this book

This is a collection of readily reproducible techniques for the study of mRNA processing and metabolism. These cutting-edge techniques range from cotranscriptional processing events that occur while the mNA is engaged with elongating RNA polymerase II, to in vivo and in vitro splicing and its biochemical analysis, and alternative splicing. Additional methods cover mRNA export, the recovery and analysis of mRNP complexes, cytoplasmic translation, mRNA degradation in vivo and in vitro, and the controversial concept of nuclear translation. Each proven protocol is described in step-by-step detail and contains a background introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

Contents

Using Chromatin Immunoprecipitation to Map Cotranscriptional mRNA Processing in Saccharomyces cerevisiae Michael-Christopher Keogh and Stephen Buratowski Identifying PhosphoCTD-Associating Proteins Hemali P. Phatnani and Arno L. Greenleaf Imaging Alternative Splicing in Living Cells Eric J. Wagner, Andrea Baines, Todd Albrecht, Robert M. Brazas, and Mariano A. Garcia-Blanco Tobramycin Affinity Tag Purification of Spliceosomes Klaus Hartmuth, Hans-Peter Vornlocher, and Reinhard Luhrmann Using Single-Strand Conformational Polymorphism Gel Electrophoresis to Analyze Mutually Exclusive Alternative Splicing Alicia M. Celotto and Brenton R. Graveley Methods for the Analysis of Adenosine-to-Inosine Editing in RNA Zuo Zhang and Gordon G. Carmichael Assaying Nuclear Messenger RNA Export in Human Cells Bryan R. Cullen RNA Unwinding Assay for DExD/H-Box RNA Helicases Stephanie S. Tseng-Rogenski and Tien-Hsien Chang Approaches for Monitoring Nuclear Translation Francisco J. Iborra, Dean A. Jackson, and Peter R. Cook Immunopurification and Analysis of Protein and RNA Components of mRNP in Mammalian Cells Fabrice Lejeune and Lynne E. Maquat Gene Expression Analysis of Messenger RNP Complexes Luiz O. F. Penalva, Scott A. Tenenbaum, and Jack D. Keene Using the lN Peptide to Tether Proteins to RNAs Julie Baron-Benhamou, Niels H. Gehring, Andreas E. Kulozik, and Matthias W. Hentze An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro Yuri V. Svitkin and Nahum Sonenberg A Poly(A) Tail-Responsive In Vitro System for Cap- or IRES-Driven Translation From HeLa Cells Christian Thoma, Antje Ostareck-Lederer, and Matthias W. Hentze Assessing Messenger RNA Decapping in Cellular Extracts Naomi Bergman, Joseph Milone, Elizabeth J. Bates, Mateusz Opyrchal, Vivian Bellofatto, and Jeffrey Wilusz Analysis of RNA Exonucleolytic Activities in Cellular Extracts Devi Mukherjee, David T. Fritz, Walter J. Kilpatrick, Min Gao, and Jeffrey Wilusz Application of Ligation-Mediated Reverse Transcription Polymerase Chain Reaction to the Identification of In Vivo Endonuclease-Generated Messenger RNA Decay Intermediates Mark N. Hanson and Daniel R. Schoenberg Biochemical Dissection of RNA Silencing in Plants Guiliang Tang and Phillip D. Zamore RNA Interference of mRNA Processing Factors in Drosophila S2 Cells Alicia M. Celotto and Brenton R. Graveley RNA Interference by Short Hairpin RNAs Expressed in Vertebrate Cells Gregory J. Hannon and Douglas S. Conklin Index

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