Advertisement

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

  • John C. Zinder
  • Christopher D. LimaEmail author
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

3′ to 5′ RNA degradation is primarily catalyzed by the RNA exosome subunits Dis3 and Rrp6 in the nucleus of Saccharomyces cerevisiae. These enzymes form a complex with the nine-subunit noncatalytic core (Exo9) to carry out their functions in vivo. Protein cofactors Rrp47, Mpp6, and the Mtr4 RNA helicase also assist the complex by modulating its activities and/or recruiting it to specific RNAs for processing or degradation. Here we present our preferred strategy for reconstituting RNA exosomes from S. cerevisiae using purified, recombinantly expressed components.

Key words

RNA exosome RNA decay Exoribonuclease Budding yeast 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported in part by GM065872 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.

References

  1. 1.
    Kilchert C, Wittmann S, Vasiljeva L (2016) The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol 17:227–239CrossRefGoogle Scholar
  2. 2.
    Łabno A, Tomecki R, Dziembowski A (2016) Cytoplasmic RNA decay pathways - enzymes and mechanisms. Biochim Biophys Acta 1863:3125–3147CrossRefGoogle Scholar
  3. 3.
    Houseley J, Tollervey D (2009) The many pathways of RNA degradation. Cell 136:763–776CrossRefGoogle Scholar
  4. 4.
    Zinder JC, Lima CD (2017) Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev 31:88–100PubMedPubMedCentralGoogle Scholar
  5. 5.
    LaCava J, Houseley J, Saveanu C et al (2005) RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121:713–724CrossRefGoogle Scholar
  6. 6.
    Wyers F, Rougemaille M, Badis G et al (2005) Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121:725–737CrossRefGoogle Scholar
  7. 7.
    Vanacova S, Wolf J, Martin G et al (2005) A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol 3:e189–e112CrossRefGoogle Scholar
  8. 8.
    Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127:1223–1237CrossRefGoogle Scholar
  9. 9.
    Bonneau F, Basquin J, Ebert J et al (2009) The yeast exosome functions as a macromolecular cage to channel RNA substrates for degradation. Cell 139:547–559CrossRefGoogle Scholar
  10. 10.
    Wasmuth EV, Lima CD (2012) Exo- and endoribonucleolytic activities of yeast cytoplasmic and nuclear RNA exosomes are dependent on the noncatalytic core and central channel. Mol Cell 48:133–144CrossRefGoogle Scholar
  11. 11.
    Makino DL, Baumgärtner M, Conti E (2013) Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495:70–75CrossRefGoogle Scholar
  12. 12.
    Wasmuth EV, Januszyk K, Lima CD (2014) Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA. Nature 511:435–439CrossRefGoogle Scholar
  13. 13.
    Wasmuth EV, Zinder JC, Zattas D et al (2017) Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase. elife 6:213CrossRefGoogle Scholar
  14. 14.
    Schuch B, Feigenbutz M, Makino DL et al (2014) The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase. EMBO J 33:2829–2846CrossRefGoogle Scholar
  15. 15.
    Falk S, Bonneau F, Ebert J et al (2017) Mpp6 incorporation in the nuclear exosome contributes to RNA channeling through the Mtr4 helicase. Cell Rep 20:2279–2286CrossRefGoogle Scholar
  16. 16.
    Weick EM, Puno MR, Januszyck K et al Helicase-dependent RNA decay illuminated by a cryo-EM structure of a human nuclear RNA exosome-MTR4 complex. Cell 173(7):1663–1667CrossRefGoogle Scholar
  17. 17.
    Schuller JM, Falk S, Fromm L et al (2018) Structure of the nuclear exosome captured on a maturing preribosome. Science 360:219–222CrossRefGoogle Scholar
  18. 18.
    Makino DL, Schuch B, Stegmann E et al (2015) RNA degradation paths in a 12-subunit nuclear exosome complex. Nature 524:54–58CrossRefGoogle Scholar
  19. 19.
    Greimann JC, Lima CD (2008) Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae: cloning, expression, purification, and activity assays. Methods Enzymol 448:185–210CrossRefGoogle Scholar
  20. 20.
    Marblestone JG, Edavettal SC, Lim Y et al (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189CrossRefGoogle Scholar
  21. 21.
    Mossessova E, Lima CD (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol Cell 5:865–876CrossRefGoogle Scholar
  22. 22.
    Wasmuth EV, Lima CD (2017) The Rrp6 C-terminal domain binds RNA and activates the nuclear RNA exosome. Nucleic Acids Res 45:846–860CrossRefGoogle Scholar
  23. 23.
    Zinder JC, Wasmuth EV, Lima CD (2016) Nuclear RNA exosome at 3.1 Å reveals substrate specificities, RNA paths, and allosteric inhibition of Rrp44/Dis3. Mol Cell 64:734–745CrossRefGoogle Scholar
  24. 24.
    Stead JA, Costello JL, Livingstone MJ, Mitchell P (2007) The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein. Nucleic Acids Res 35:5556–5567CrossRefGoogle Scholar
  25. 25.
    Dedic E, Seweryn P, Jonstrup AT et al (2014) Structural analysis of the yeast exosome Rrp6p-Rrp47p complex by small-angle X-ray scattering. Biochem Biophys Res Commun 450:634–640CrossRefGoogle Scholar
  26. 26.
    Feigenbutz M, Garland W, Turner M, Mitchell P (2013) The exosome cofactor Rrp47 is critical for the stability and normal expression of its associated exoribonuclease Rrp6 in Saccharomyces cerevisiae. PLoS One 8:e80752CrossRefGoogle Scholar
  27. 27.
    Lebreton A, Tomecki R, Dziembowski A, Séraphin B (2008) Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature 456:993–996CrossRefGoogle Scholar
  28. 28.
    Wang H-W, Wang J, Ding F et al (2007) Architecture of the yeast Rrp44 exosome complex suggests routes of RNA recruitment for 3′ end processing. Proc Natl Acad Sci U S A 104:16844–16849CrossRefGoogle Scholar
  29. 29.
    Liu J-J, Niu C-Y, Wu Y et al (2016) CryoEM structure of yeast cytoplasmic exosome complex. Cell Res 26:822–837CrossRefGoogle Scholar
  30. 30.
    Kowalinski E, Kögel A, Ebert J et al (2016) Structure of a cytoplasmic 11-subunit RNA exosome complex. Mol Cell 63:125–134CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Tri-Institutional Training Program in Chemical BiologyMemorial Sloan Kettering Cancer CenterNew YorkUSA
  2. 2.Structural Biology Program, Sloan Kettering InstituteMemorial Sloan Kettering Cancer CenterNew YorkUSA
  3. 3.Howard Hughes Medical InstituteNew YorkUSA

Personalised recommendations

Cite protocol

Buy options