mRNA Processing and Metabolism

Cells possess a wealth of posttranscriptional control mechanisms that impact on every conceivable aspect of the life of an mRNA. These processes are intimately intertwined in an almost baroque manner, where promoter context influences the recruitment of splicing factors, where the majority of pre-mRNAs undergo alternative splicing, and where proteins deposited during nuclear processing impact distal cytoplasmic processing, translation, and decay. If there is a unifying theme to mRNA Processing and Metabolism: Methods and Protocols, it is that mRNA processing and metabolism are integrated processes. Many of the techniques used to study mRNA have been described in a previous volume of this series (RNAProtein Interaction Protocols, Susan Haynes, ed.) and specialized methods journals. In selecting topics for mRNA Processing and Metabolism: Methods and Protocols, I sought input on new and novel techniques and approaches that build on this foundation using technological advances in microscopy, whole genome sequencing, microarrays, mass spectrometry, fluorescent detection methodologies, and RNA interference. I have tried not to bias this book toward any single model organism, and approaches described in the various chapters use yeast, Drosophila, Xenopus, mice, plants, and cultured mammalian cells.

Includes supplementary material: sn.pub/extras

Klappentext
mRNA processing is a key step in gene expression that effects all the proteins within the cell. In mRNA Processing and Metabolism: Methods and Protocols, world-renowned researchers bring together the latest techniques spanning the breadth of mRNA processing and metabolism. Drawing on recent advances in microscopy, whole genome sequencing, microarrays, mass spectrometry, fluorescent detection methodologies, and RNA interference, the authors offer readily reproducible methods for the cotranscriptional processing events that occur while the mRNA is engaged with elongating RNA polymerase II, with splicing and its biochemical analysis and with 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. A variety of model organisms are used, including yeast, Drosophila, Xenopus, mice, plants, and cultured mammalian cells. Each proven protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Timely and authoritative, mRNA Processing and Metabolism: Methods and Protocols provides a powerful collection of novel techniques that are not only effective, but also immediately applicable to current problems in many areas of biological research.

Inhalt
Using Chromatin Immunoprecipitation to Map Cotranscriptional mRNA Processing in Saccharomyces cerevisiae.- Identifying PhosphoCTD-Associating Proteins.- Imaging Alternative Splicing in Living Cells.- Tobramycin Affinity Tag Purification of Spliceosomes.- Using Single-Strand Conformational Polymorphism Gel Electrophoresis to Analyze Mutually Exclusive Alternative Splicing.- Methods for the Analysis of Adenosine-to-Inosine Editing in RNA.- Assaying Nuclear Messenger RNA Export in Human Cells.- RNA Unwinding Assay for DExD/H-Box RNA Helicases.- Approaches for Monitoring Nuclear Translation.- Immunopurification and Analysis of Protein and RNA Components of mRNP in Mammalian Cells.- Gene Expression Analysis of Messenger RNP Complexes.- Using the ?N Peptide to Tether Proteins to RNAs.- An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro.- A Poly(A) Tail-Responsive In Vitro System for Cap- or IRES-Driven Translation From HeLa Cells.- Assessing Messenger RNA Decapping in Cellular Extracts.- Analysis of RNA Exonucleolytic Activities in Cellular Extracts.- Application of Ligation-Mediated Reverse Transcription Polymerase Chain Reaction to the Identification of In Vivo Endonuclease-Generated Messenger RNA Decay Intermediates.- Biochemical Dissection of RNA Silencing in Plants.- RNA Interference of mRNA Processing Factors in Drosophila S2 Cells.- RNA Interference by Short Hairpin RNAs Expressed in Vertebrate Cells.