Advertisement

Purification of Eukaryotic Exoribonucleases Following Heterologous Expression in Bacteria and Analysis of Their Biochemical Properties by In Vitro Enzymatic Assays

  • Rafal TomeckiEmail author
  • Karolina Drazkowska
  • Antonina Krawczyk
  • Katarzyna Kowalska
  • Andrzej DziembowskiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

Exoribonucleases—among the other RNases—play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specificity and optimal conditions for enzymatic action. One of the most significant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5′-end to 3′-end or in the opposite way. Here, we present methods allowing the efficient production and purification of eukaryotic exoribonucleases, the preparation and labeling of various RNA substrates, and the biochemical characterization of exonucleolytic activity. We also explain how the exonucleolytic activity may be distinguished from that of endonucleases.

Key words

Exoribonuclease RNase RNA degradation RNA Oligonucleotide labeling Isotope Fluorescent dye Enzymatic activity Thin-layer chromatography Denaturing polyacrylamide gel 

Notes

Acknowledgments

This work was supported by the National Science Centre within the grant allocated to RT on the basis of the decision number DEC-2011/01/D/NZ1/03510 and through a grant awarded to RT by the National Centre for Research and Development within the LIDER program (LIDER/35/46/L-3/11/NCBR/2012). RT was the recipient of a scholarship for outstanding young scientists from the Polish Ministry of Science and Higher Education.

References

  1. 1.
    Houseley J, Tollervey D (2009) The many pathways of RNA degradation. Cell 136:763–776PubMedCrossRefGoogle Scholar
  2. 2.
    Stoecklin G, Mühlemann O (2013) RNA decay mechanisms: specificity through diversity. Biochim Biophys Acta 1829:487–490PubMedCrossRefGoogle Scholar
  3. 3.
    Tomecki R, Drazkowska K, Dziembowski A (2010) Mechanisms of RNA degradation by the eukaryotic exosome. Chembiochem 11:938–945PubMedCrossRefGoogle Scholar
  4. 4.
    Tomecki R, Kristiansen MS, Lykke-Andersen S et al (2010) The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L. EMBO J 29:2342–2357PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Staals RH, Bronkhorst AW, Schilders G et al (2010) Dis3-like 1: a novel exoribonuclease associated with the human exosome. EMBO J 29:2358–2367PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Lykke-Andersen S, Tomecki R, Jensen TH et al (2011) The eukaryotic RNA exosome: same scaffold but variable catalytic subunits. RNA Biol 8:61–66PubMedCrossRefGoogle Scholar
  7. 7.
    Bonneau F, Basquin J, Ebert J et al (2009) The yeast exosome functions as a macromolecular cage to channel RNA substrates for degradation. Cell 139:547–559PubMedCrossRefGoogle Scholar
  8. 8.
    Malet H, Topf M, Clare DK et al (2010) RNA channelling by the eukaryotic exosome. EMBO Rep 11:936–942PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    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 48:133–144PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Drazkowska K, Tomecki R, Stodus K et al (2013) The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro. Nucleic Acids Res 41:3845–3858PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127:1223–1237PubMedCrossRefGoogle Scholar
  12. 12.
    Dziembowski A, Lorentzen E, Conti E et al (2007) A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol Biol 14:15–22PubMedCrossRefGoogle Scholar
  13. 13.
    Lorentzen E, Basquin J, Tomecki R et al (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 29:717–728PubMedCrossRefGoogle Scholar
  14. 14.
    Lebreton A, Tomecki R, Dziembowski A et al (2008) Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature 456:993–996PubMedCrossRefGoogle Scholar
  15. 15.
    Schaeffer D, Tsanova B, Barbas A et al (2009) The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities. Nat Struct Mol Biol 16:56–62PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Schneider C, Leung E, Brown J et al (2009) The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome. Nucleic Acids Res 37:1127–1140PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Nagarajan VK, Jones CI, Newbury SF et al (2013) XRN 5′→3′ exoribonucleases: structure, mechanisms and functions. Biochim Biophys Acta 1829:590–603PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Jinek M, Coyle SM, Doudna JA (2011) Coupled 5′ nucleotide recognition and processivity in Xrn1-mediated mRNA decay. Mol Cell 41:600–608PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Braun JE, Truffault V, Boland A et al (2012) A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5′ exonucleolytic degradation. Nat Struct Mol Biol 19:1324–1331PubMedCrossRefGoogle Scholar
  20. 20.
    Pellegrini O, Mathy N, Condon C et al (2008) In vitro assays of 5′ to 3′-exoribonuclease activity. Methods Enzymol 448:167–183PubMedCrossRefGoogle Scholar
  21. 21.
    Tomecki R, Drazkowska K, Kucinski I et al (2014) Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target. Nucleic Acids Res 42:1270–1290PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Clouet-d’Orval B, Rinaldi D, Quentin Y et al (2010) Euryarchaeal beta-CASP proteins with homology to bacterial RNase J have 5′- to 3′-exoribonuclease activity. J Biol Chem 285:17574–17583PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Rafal Tomecki
    • 1
    • 2
    Email author
  • Karolina Drazkowska
    • 1
    • 2
  • Antonina Krawczyk
    • 2
    • 3
  • Katarzyna Kowalska
    • 1
    • 2
  • Andrzej Dziembowski
    • 1
    • 2
    Email author
  1. 1.Institute of Biochemistry and BiophysicsPolish Academy of SciencesWarsawPoland
  2. 2.Institute of Genetics and Biotechnology, Faculty of BiologyUniversity of WarsawWarsawPoland
  3. 3.Department of Molecular GeneticsGroningen Biomolecular Sciences and Biotechnology Institute, University of GroningenGroningenThe Netherlands

Personalised recommendations