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In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes

  • Alexandra Zinoviev
  • Christopher U. T. Hellen
  • Tatyana V. PestovaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

The RNA exosome is a multisubunit protein complex that exhibits a 3′ to 5′ exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.

Key words

RNA exosome Cap labeling of mRNA In vitro reconstitution of mammalian ribosomal complexes Toeprinting GTPBP1 mRNA degradation assay 

Notes

Acknowledgments

This work was supported by NIH grant GM80623 to T.V.P. and NIH grant AI123406 to C.U.T.H.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Alexandra Zinoviev
    • 1
  • Christopher U. T. Hellen
    • 1
  • Tatyana V. Pestova
    • 1
    Email author
  1. 1.Department of Cell BiologySUNY Downstate Health Sciences UniversityBrooklynUSA

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