Skip to main content
SpringerLink
Log in

Cryo-Electron Microscopy of Endogenous Yeast Exosomes

  • Protocol
  • First Online:
Book cover The Eukaryotic RNA Exosome

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

Abstract

The RNA exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3′-to-5′ exo- and endo-RNase activities. With assistance from nuclear or cytoplasmic cofactors, functional studies implicated the exosome as a critical player in the turnover of almost all RNA species, including mRNAs, rRNA, tRNAs, and other noncoding RNAs. Here, we describe the purification of the yeast 10-subunit exosome and 11-subunit Exosome-Ski7, as well as subsequent sample screening by negative staining EM and structural analysis by cryo-EM.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Doma MK, Parker R (2007) RNA quality control in eukaryotes. Cell 131(4):660–668

    Article  CAS  PubMed  Google Scholar 

  2. Mitchell P, Petfalski E, Shevchenko A, Mann M, Tollervey D (1997) The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→ 5′ exoribonucleases. Cell 91(4):457–466

    Article  CAS  PubMed  Google Scholar 

  3. Garneau NL, Wilusz J, Wilusz CJ (2007) The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 8(2):113

    Article  CAS  PubMed  Google Scholar 

  4. Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127(6):1223–1237

    Article  CAS  PubMed  Google Scholar 

  5. Lorentzen E, Basquin J, Tomecki R, Dziembowski A, Conti E (2008) Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family. Mol Cell 29(6):717–728

    Article  CAS  PubMed  Google Scholar 

  6. Wang H-W, Wang J, Ding F, Callahan K, Bratkowski MA, Butler JS, Nogales E, Ke A (2007) Architecture of the yeast Rrp44–exosome complex suggests routes of RNA recruitment for 3′ end processing. Proc Natl Acad Sci 104(43):16844–16849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Makino DL, Baumgärtner M, Conti E (2013) Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495(7439):70

    Article  CAS  PubMed  Google Scholar 

  8. Liu J-J, Bratkowski MA, Liu X, Niu C-Y, Ke A, Wang H-W (2014) Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM. Nat Struct Mol Biol 21(1):95

    Article  CAS  PubMed  Google Scholar 

  9. Butler JS, Mitchell P (2010) Rrp6, rrp47 and cofactors of the nuclear exosome. In: RNA Exosome. Springer, pp 91–104

    Google Scholar 

  10. Wasmuth EV, Januszyk K, Lima CD (2014) Structure of an Rrp6–RNA exosome complex bound to poly (A) RNA. Nature 511(7510):435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schaeffer D, Clark A, Klauer AA, Tsanova B, van Hoof A (2010) Functions of the cytoplasmic exosome. In: RNA exosome. Springer, pp 79–90

    Google Scholar 

  12. Araki Y, Takahashi S, Kobayashi T, Kajiho H, Hoshino S, Katada T (2001) Ski7p G protein interacts with the exosome and the Ski complex for 3′-to-5′ mRNA decay in yeast. EMBO J 20(17):4684–4693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Synowsky SA, van Wijk M, Raijmakers R, Heck AJ (2009) Comparative multiplexed mass spectrometric analyses of endogenously expressed yeast nuclear and cytoplasmic exosomes. J Mol Biol 385(4):1300–1313

    Article  CAS  PubMed  Google Scholar 

  14. Shi Y, Pellarin R, Fridy PC, Fernandez-Martinez J, Thompson MK, Li Y, Wang QJ, Sali A, Rout MP, Chait BT (2015) A strategy for dissecting the architectures of native macromolecular assemblies. Nat Methods 12(12):1135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu J-J, Niu C-Y, Wu Y, Tan D, Wang Y, Ye M-D, Liu Y, Zhao W, Zhou K, Liu Q-S (2016) CryoEM structure of yeast cytoplasmic exosome complex. Cell Res 26(7):822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116(1):190–199

    Article  CAS  PubMed  Google Scholar 

  17. van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116(1):17–24

    Article  PubMed  Google Scholar 

  18. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128(1):82–97

    Article  CAS  PubMed  Google Scholar 

  19. Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157(1):38–46

    Article  CAS  PubMed  Google Scholar 

  20. Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180(3):519–530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612

    Article  CAS  PubMed  Google Scholar 

  22. Liu X, Wang H-W (2011) Single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method. J Vis Exp 49

    Google Scholar 

  23. McDowall A, Dobro M, Melanson L, Jensen G (2010) Cryo-EM part A: sample preparation and data collection. Methods Enzymol 481:63–82

    PubMed  Google Scholar 

  24. Atlas RM (2010) Handbook of microbiological media. CRC Press, Boca Raton

    Book  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jun-Jie Liu

  2. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Jun-Jie Liu

  3. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Hong-Wei Wang

Authors
  1. Jun-Jie Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hong-Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jun-Jie Liu or Hong-Wei Wang .

Editor information

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

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Liu, JJ., Wang, HW. (2020). Cryo-Electron Microscopy of Endogenous Yeast Exosomes. 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_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9822-7_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9821-0

  • Online ISBN: 978-1-4939-9822-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation