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Enzymatic Analysis of Reconstituted Archaeal Exosomes

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The Eukaryotic RNA Exosome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2062))

Abstract

The archaeal exosome is a protein complex with phosphorolytic activity. It is built of a catalytically active hexameric ring containing the archaeal Rrp41 and Rrp42 proteins, and a heteromeric RNA-binding platform. The platform contains a heterotrimer of the archaeal Rrp4 and Csl4 proteins (which harbor S1 and KH or Zn-ribbon RNA binding domains), and comprises additional archaea-specific subunits. The latter are represented by the archaeal DnaG protein, which harbors a novel RNA-binding domain and tightly interacts with the majority of the exosome isoforms, and Nop5, known as a part of an rRNA methylating complex and found to associate with the archaeal exosome at late stationary phase. Although in the cell the archaeal exosome exists in different isoforms with heterotrimeric Rrp4-Csl4-caps, in vitro it is possible to reconstitute complexes with defined, homotrimeric caps and to study the impact of each RNA-binding subunit on exoribonucleolytic degradation and on polynucleotidylation of RNA. Here we describe procedures for reconstitution of isoforms of the Sulfolobus solfataricus exosome and for set-up of RNA degradation and polyadenylation assays.

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Authors and Affiliations

  1. Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany

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

Authors
  1. Elena Evguenieva-Hackenberg
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  2. A. Susann Gauernack
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  3. Linlin Hou
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  4. Gabriele Klug
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Corresponding authors

Correspondence to Elena Evguenieva-Hackenberg or Gabriele Klug .

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Editors and Affiliations

  1. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA

    John LaCava

  2. Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

    Štěpánka Vaňáčová

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Evguenieva-Hackenberg, E., Gauernack, A.S., Hou, L., Klug, G. (2020). Enzymatic Analysis of Reconstituted Archaeal Exosomes. In: LaCava, J., Vaňáčová, Š. (eds) The Eukaryotic RNA Exosome. Methods in Molecular Biology, vol 2062. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9822-7_4

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  • DOI: https://doi.org/10.1007/978-1-4939-9822-7_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9821-0

  • Online ISBN: 978-1-4939-9822-7

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