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Reconstitution of the Schizosaccharomyces pombe RNA Exosome

  • Kurt Januszyk
  • Christopher D. LimaEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

RNA exosome RNA decay Ribonuclease Helicase Fission yeast 

Notes

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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Copyright information

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

Authors and Affiliations

  1. 1.Structural Biology Program, Sloan Kettering InstituteMemorial Sloan Kettering Cancer CenterNew YorkUSA
  2. 2.Howard Hughes Medical InstituteNew YorkUSA

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