The Eukaryotic RNA Exosome

This volume provides a cross-section of RNA exosome research protocols, applied to a diversity of model organisms. Chapters guide readers through methods that e.g. delineate eukaryotic exosomes' origins in prokaryotes, probe its RNA substrates, adapter complexes and macromolecular interaction of networks, and establish critical structural-function relationships. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, The Eukaryotic RNA Exosome: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

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Contenu

The RNA Exosome and Human Disease.- The Bacterial Counterparts of the Eukaryotic Exosome: An Evolutionary Perspective.- In vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex.- Enzymatic Analysis of Reconstituted Archaeal Exosomes.- Protocols for Northern Analysis of Exosome Substrates and Other Non-coding RNAs.- Mapping Exosome-substrate Interactions in vivo by UV-crosslinking.- Global Identification of Human Exosome Substrates using RNA Interference and RNA-sequencing.- High-resolution Mapping of 3' Extremities of RNA Exosome Substrates by 3' RACE-seq.- Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion.-Thiouridine-to-Cytidine Conversion Sequencing (TUC-seq) to Measure mRNA Transcription and Degradation Rates.- RNA Exosomes and their Co-factors.- Purification of Endogenous Tagged TRAMP4/5 and Exosome Complexes from Yeast and in vitro Polyadenylation-Exosome Activation Assays.- Comparative poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants.- Purification and in vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2.- Affinity Proteomic Analysis of the Human Exosome and its Cofactor Complexes.- In vitro Characterization of the Activity of the Mammalian RNA exosome on mRNAs in Ribosomal Translation Complexes.- Native Mass Spectrometry Analysis of Affinity-captured Endogenous Yeast RNA Exosome Complexes.- Chemical Cross-Linking and Mass Spectrometric Analysis of the Endogenous Yeast Exosome Complexes.- Cryo-Electron Microscopy of Endogenous Yeast Exosomes.- Strategies for Generating RNA Exosome Complexes from Recombinant Expression Hosts.- Reconstitution of S. cerevisiae RNA Exosome Complexes Using Recombinantly Expressed Proteins.- Reconstitution of the Schizosaccharomyces pombe RNA Exosome.- Reconstitution of the Human Nuclear RNA Exosome.- Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies.