Reconstitution of the Schizosaccharomyces pombe RNA Exosome

  • Kurt Januszyk
  • Christopher D. LimaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)


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 



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.


  1. 1.
    Zinder JC, Lima CD (2017) Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev 31(2):88–100CrossRefGoogle Scholar
  2. 2.
    Januszyk K, Lima CD (2014) The eukaryotic RNA exosome. Curr Opin Struct Biol 24:132–140CrossRefGoogle Scholar
  3. 3.
    Januszyk K, Lima CD (2011) Structural components and architectures of RNA exosomes. Adv Exp Med Biol 702:9–28CrossRefGoogle 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(4):227–239CrossRefGoogle Scholar
  5. 5.
    Zofall M, Grewal SI (2006) RNAi-mediated heterochromatin assembly in fission yeast. Cold Spring Harb Symp Quant Biol 71:487–496CrossRefGoogle Scholar
  6. 6.
    Grewal SI (2010) RNAi-dependent formation of heterochromatin and its diverse functions. Curr Opin Genet Dev 20(2):134–141CrossRefGoogle Scholar
  7. 7.
    Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16(2):71–84CrossRefGoogle Scholar
  8. 8.
    Buhler M, Moazed D (2007) Transcription and RNAi in heterochromatic gene silencing. Nat Struct Mol Biol 14(11):1041–1048CrossRefGoogle Scholar
  9. 9.
    Lemay JF, Larochelle M, Marguerat S, Atkinson S, Bahler J, Bachand F (2014) The RNA exosome promotes transcription termination of backtracked RNA polymerase II. Nat Struct Mol Biol 21(10):919–926CrossRefGoogle Scholar
  10. 10.
    Sugiyama T, Thillainadesan G, Chalamcharla VR, Meng Z, Balachandran V, Dhakshnamoorthy J, Zhou M, Grewal SIS (2016) Enhancer of rudimentary cooperates with conserved RNA-processing factors to promote meiotic mRNA decay and facultative heterochromatin assembly. Mol Cell 61(5):747–759CrossRefGoogle Scholar
  11. 11.
    Lee NN, Chalamcharla VR, Reyes-Turcu F, Mehta S, Zofall M, Balachandran V, Dhakshnamoorthy J, Taneja N, Yamanaka S, Zhou M, Grewal SI (2013) Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance. Cell 155(5):1061–1074CrossRefGoogle Scholar
  12. 12.
    Egan ED, Braun CR, Gygi SP, Moazed D (2014) Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex. RNA 20(6):867–881CrossRefGoogle Scholar
  13. 13.
    Wasmuth EV, Zinder JC, Zattas D, Das M, Lima CD (2017) Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase. Elife 6Google Scholar
  14. 14.
    Schuch B, Feigenbutz M, Makino DL, Falk S, Basquin C, Mitchell P, Conti E (2014) The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase. EMBO J 33(23):2829–2846CrossRefGoogle Scholar
  15. 15.
    Milligan L, Decourty L, Saveanu C, Rappsilber J, Ceulemans H, Jacquier A, Tollervey D (2008) A yeast exosome cofactor, Mpp6, functions in RNA surveillance and in the degradation of noncoding RNA transcripts. Mol Cell Biol 28(17):5446–5457CrossRefGoogle Scholar
  16. 16.
    Garland W, Feigenbutz M, Turner M, Mitchell P (2013) Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6. RNA 19(12):1659–1668CrossRefGoogle Scholar
  17. 17.
    Buhler M, Spies N, Bartel DP, Moazed D (2008) TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway. Nat Struct Mol Biol 15(10):1015–1023CrossRefGoogle Scholar
  18. 18.
    Weick EM, Puno MR, Januszyk K, Zinder JC, DiMattia MA, Lima CD (2018) Helicase-dependent RNA decay illuminated by a cryo-EM structure of a human nuclear RNA exosome-MTR4 complex. Cell 173(7): 1663–1677CrossRefGoogle Scholar
  19. 19.
    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(5):865–876CrossRefGoogle Scholar
  20. 20.
    Wood V, Harris MA, McDowall MD, Rutherford K, Vaughan BW, Staines DM, Aslett M, Lock A, Bahler J, Kersey PJ, Oliver SG (2012) PomBase: a comprehensive online resource for fission yeast. Nucleic Acids Res 40:D695–D699CrossRefGoogle Scholar
  21. 21.
    McDowall MD, Harris MA, Lock A, Rutherford K, Staines DM, Bahler J, Kersey PJ, Oliver SG, Wood V (2015) PomBase 2015: updates to the fission yeast database. Nucleic Acids Res 43:D656–D661CrossRefGoogle Scholar
  22. 22.
    Zinder JC, Wasmuth EV, Lima CD (2016) Nuclear RNA exosome at 3.1 A reveals substrate specificities, RNA paths, and allosteric inhibition of Rrp44/Dis3. Mol Cell 64(4):734–745CrossRefGoogle Scholar
  23. 23.
    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(1):133–144CrossRefGoogle Scholar
  24. 24.
    Wasmuth EV, Januszyk K, Lima CD (2014) Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA. Nature 511(7510):435–439CrossRefGoogle Scholar
  25. 25.
    Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127(6):1223–1237CrossRefGoogle Scholar
  26. 26.
    Feigenbutz M, Jones R, Besong TM, Harding SE, Mitchell P (2013) Assembly of the yeast exoribonuclease Rrp6 with its associated cofactor Rrp47 occurs in the nucleus and is critical for the controlled expression of Rrp47. J Biol Chem 288(22):15959–15970CrossRefGoogle Scholar
  27. 27.
    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(11):e80752CrossRefGoogle Scholar
  28. 28.
    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(16):5556–5567CrossRefGoogle Scholar

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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