Main description:

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.

Contents:

Part I: Introduction to the RNA Exosome in Biomedicine

1. The RNA Exosome and Human Disease

Milo B. Fasken, Derrick J. Morton, Emily G. Kuiper, Stephanie K. Jones, Sara W. Leung, and Anita H. Corbett

Part II: Prokaryotic RNases and Exosomes

2. The Bacterial Counterparts of the Eukaryotic Exosome: An Evolutionary Perspective

Sandra C. Viegas , Rute G. Matos, and Cecilia M. Arraiano

3. In vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex

Rute G. Matos, Sandra C. Viegas, and Cecilia M. Arraiano

4. Enzymatic Analysis of Reconstituted Archaeal Exosomes

Elena Evguenieva-Hackenberg, A. Susann Gauernack, Linlin Hou, and Gabriele Klug

Part III: Analyzing Exosome Substrates

5. Protocols for Northern Analysis of Exosome Substrates and Other Non-coding RNAs

Cristina Cruz and Jonathan Houseley

6. Mapping Exosome-substrate Interactions in vivo by UV-crosslinking

Clementine Delan-Forino and David Tollervey

7. Global Identification of Human Exosome Substrates using RNA Interference and RNA-sequencing

Marta Lloret-Llinares and Torben Heick Jensen

8. High-resolution Mapping of 3' Extremities of RNA Exosome Substrates by 3' RACE-seq

Helene Scheer, Caroline De Almeida, Natalia Sikorska, Sandrine Koechler, Dominique Gagliardi, and Helene Zuber

9. Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion

Veronika A. Herzog, Nina Fasching, and Stefan L. Ameres

10. Thiouridine-to-Cytidine Conversion Sequencing (TUC-seq) to Measure mRNA Transcription and Degradation Rates

Alexandra Lusser, Catherina Gasser, Lukas Trixl, Paolo Piatti, Isabel Delazer, Dietmar Rieder, Jeffrey Bashin, Christian Riml, Thomas Amort, and Ronald Micura

Part IV: Exosomes and their Supramolecular Functional Interactions

11. RNA Exosomes and their Co-factors

Cornelia Kilchert

12. Purification of Endogenous Tagged TRAMP4/5 and Exosome Complexes from Yeast and in vitro Polyadenylation-Exosome Activation Assays

Dagmar Zigackova, Veronika Rajecka, and Stepanka Vanacova

13. Comparative poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants

Cornelia Kilchert, Svenja Hester, Alfredo Castello, Shabaz Mohammed, and Lidia Vasiljeva

14. Purification and in vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2

Odil Porrua

15. Affinity Proteomic Analysis of the Human Exosome and its Cofactor Complexes

Kinga Winczura, Michal Domanski, and John LaCava

16. In vitro Characterization of the Activity of the Mammalian RNA exosome on mRNAs

in Ribosomal Translation Complexes

Alexandra Zinoviev, Christopher U.T. Hellen, and Tatyana V. Pestova

Part V: Recombinant Expression Strategies and Structural Characterization of Exosomes

17. Native Mass Spectrometry Analysis of Affinity-captured Endogenous Yeast RNA Exosome Complexes

Paul Dominic B. Olinares and Brian T. Chait

18. Chemical Cross-Linking and Mass Spectrometric Analysis of the Endogenous Yeast Exosome Complexes

Yufei Xiang, Zhuolun Shen, and Yi Shi

19. Cryo-Electron Microscopy of Endogenous Yeast Exosomes

Jun-Jie Liu and Hong-Wei Wang

20. Strategies for Generating RNA Exosome Complexes from Recombinant Expression Hosts

Eva-Maria Weick, John C. Zinder, and Christopher D. Lima

21. Reconstitution of S. cerevisiae RNA Exosome Complexes Using Recombinantly Expressed Proteins

John C. Zinder and Christopher D. Lima

22. Reconstitution of the Schizosaccharomyces pombe RNA Exosome

Kurt Januszyk and Christopher D. Lima

23. Reconstitution of the Human Nuclear RNA Exosome

Kurt Januszyk, Eva-Maria Weick, and Christopher D. Lima

24. Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies

Achim Keidel, Elena Conti, and Sebastian Falk