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Reconstitution of the Schizosaccharomyces pombe RNA Exosome

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The Eukaryotic RNA Exosome

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

Abstract

In this chapter, we describe methods to clone, express, purify, and reconstitute active S. pombe RNA exosomes. Reconstitution procedures are similar to methods that have been successful for the human and budding yeast exosome systems using protein subunits purified from the recombinant host E. coli. By applying these strategies, we can successfully reconstitute the S. pombe noncatalytic exosome core as well as complexes that contain the exoribonucleases Dis3 and Rrp6, cofactors Cti1 (equivalent to budding yeast Rrp47) and Mpp6 as well as the RNA helicase Mtr4.

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Acknowledgments

We thank Lima Lab members for advice during the course of this work and Fangyu Liu for her contributions to reconstituting S. pombe exosomes. This work was supported in part by GM079196 and GM118080 (NIH/NIGMS, C.D.L) and P30CA008748 (NIH/National Cancer Institute). The content is the authors’ responsibility and does not represent the official views of the NIH. C.D.L is a Howard Hughes Medical Institute Investigator.

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Authors and Affiliations

  1. Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Kurt Januszyk & Christopher D. Lima

  2. Howard Hughes Medical Institute, New York, NY, USA

    Christopher D. Lima

Authors
  1. Kurt Januszyk
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  2. Christopher D. Lima
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Correspondence to Christopher D. Lima .

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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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Januszyk, K., Lima, C.D. (2020). Reconstitution of the Schizosaccharomyces pombe RNA Exosome. 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_22

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  • DOI: https://doi.org/10.1007/978-1-4939-9822-7_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9821-0

  • Online ISBN: 978-1-4939-9822-7

  • eBook Packages: Springer Protocols

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