Advertisement

Purification and Reconstitution of the S. cerevisiae TRAMP and Ski Complexes for Biochemical and Structural Studies

  • Achim Keidel
  • Elena ContiEmail author
  • Sebastian FalkEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

The RNA exosome is a macromolecular machine that degrades a large variety of RNAs from their 3′-end. It comprises the major 3′-to-5′ exonuclease in the cell, completely degrades erroneous and overly abundant RNAs, and is also involved in the precise processing of RNAs. To degrade transcripts both specifically and efficiently the exosome functions together with compartment-specific cofactors. In the yeast S. cerevisiae, the exosome associates with the Ski complex in the cytoplasm and with Mtr4 alone or with Mtr4 as part of the TRAMP complex in the nucleus. Here we describe how to produce, purify, and assemble the Ski and TRAMP complexes from S. cerevisiae.

Key words

Exosome Mtr4 Trf4 Air2 TRAMP Ski2 Ski3 Ski8 Ski complex 

References

  1. 1.
    Zinder JC, Lima CD (2017) Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev 31:88–100CrossRefGoogle Scholar
  2. 2.
    Chlebowski A, Lubas M, Jensen TH, Dziembowski A (2013) RNA decay machines: the exosome. Biochim Biophys Acta 1829:552–560PubMedPubMedCentralGoogle Scholar
  3. 3.
    Makino DL, Schuch B, Stegmann E et al (2015) RNA degradation paths in a 12-subunit nuclear exosome complex. Nature 524:54–58CrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Johnson SJ, Jackson RN (2013) Ski2-like RNA helicase structures common themes and complex assemblies. RNA Biol 10:33–43CrossRefGoogle Scholar
  6. 6.
    Weir JR, Bonneau F, Hentschel J, Conti E (2010) Structural analysis reveals the characteristic features of Mtr4, a DExH helicase involved in nuclear RNA processing and surveillance. Proc Natl Acad Sci U S A 107:12139–12144CrossRefGoogle Scholar
  7. 7.
    Jackson RN, Klauer AA, Hintze BJ et al (2010) The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing. EMBO J 29:2205–2216CrossRefGoogle Scholar
  8. 8.
    Halbach F, Rode M, Conti E (2012) The crystal structure of S. cerevisiae Ski2, a DExH helicase associated with the cytoplasmic functions of the exosome. RNA 18:124–134CrossRefGoogle Scholar
  9. 9.
    Thoms M, Thomson E, Bassler J et al (2015) The exosome is recruited to RNA substrates through specific adaptor proteins. Cell 162:1029–1038CrossRefGoogle Scholar
  10. 10.
    Falk S, Tants J-N, Basquin J et al (2017) Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53. RNA 23:1780–1787CrossRefGoogle Scholar
  11. 11.
    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
  12. 12.
    Vaňáčová Š, Wolf J, Martin G et al (2005) A new yeast poly(a) polymerase complex involved in RNA quality control. PLoS Biol 3:e189CrossRefGoogle Scholar
  13. 13.
    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
  14. 14.
    Hamill S, Wolin SL, Reinisch KM (2010) Structure and function of the polymerase core of TRAMP, a RNA surveillance complex. Proc Natl Acad Sci 107:15045–15050CrossRefGoogle Scholar
  15. 15.
    Fasken MB, Leung SW, Banerjee A et al (2011) Air1 zinc knuckles 4 and 5 and a conserved IWRXY motif are critical for the function and integrity of the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) RNA quality control complex. J Biol Chem 286:37429–37445CrossRefGoogle Scholar
  16. 16.
    Schmidt K, Butler JS (2013) Nuclear RNA surveillance: role of TRAMP in controlling exosome specificity. WIREs RNA 4:217–231CrossRefGoogle Scholar
  17. 17.
    Schmidt K, Xu Z, Mathews DH, Butler JS (2012) Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance. RNA 18:1934–1945CrossRefGoogle Scholar
  18. 18.
    Holub P, Lalakova J, Cerna H et al (2012) Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation. Nucleic Acids Res 40:5679–5693CrossRefGoogle Scholar
  19. 19.
    Falk S, Weir JR, Hentschel J et al (2014) The molecular architecture of the TRAMP complex reveals the organization and interplay of its two catalytic activities. Mol Cell 55:856–867CrossRefGoogle Scholar
  20. 20.
    Anderson JS, Parker RP (1998) The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3″ to 5″ exonucleases of the exosome complex. EMBO J 17:1497–1506CrossRefGoogle Scholar
  21. 21.
    Brown JT, Bai X, Johnson AW (2000) The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo. RNA 6:449–457CrossRefGoogle Scholar
  22. 22.
    Synowsky SA, Heck AJR (2008) The yeast Ski complex is a hetero-tetramer. Protein Sci 17:119–125CrossRefGoogle Scholar
  23. 23.
    Halbach F, Reichelt P, Rode M, Conti E (2013) The yeast Ski complex: crystal structure and RNA channeling to the exosome complex. Cell 154:814–826CrossRefGoogle Scholar
  24. 24.
    Berger I, Fitzgerald DJ, Richmond TJ (2004) Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22:1583–1587CrossRefGoogle Scholar
  25. 25.
    Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37:49–57CrossRefGoogle Scholar
  26. 26.
    Geneva Biotech (2018) Multibac™ User Manual 6.1. Accessed 3 Aug 2018Google Scholar
  27. 27.
    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
  28. 28.
    Schuller JM, Falk S, Fromm L et al (2018) Structure of the nuclear exosome captured on a maturing preribosome. Science 360:219–222CrossRefGoogle Scholar
  29. 29.
    Schmidt C, Kowalinski E, Shanmuganathan V et al (2016) The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Science 354:1431–1433CrossRefGoogle Scholar
  30. 30.
    Cheng Z, Liu Y, Wang C et al (2004) Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination. Protein Sci 13:2673–2684CrossRefGoogle Scholar
  31. 31.
    Madrona AY, Wilson DK (2004) The structure of Ski8p, a protein regulating mRNA degradation: implications for WD protein structure. Protein Sci 13:1557–1565CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Structural Cell BiologyMax-Planck-Institute of BiochemistryMartinsriedGermany
  2. 2.Max Perutz Laboratories, Department of Structural and Computational BiologyUniversity of ViennaViennaAustria

Personalised recommendations