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.
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References
Houseley J, Tollervey D (2009) The many pathways of RNA degradation. Cell 136:763–776
Stoecklin G, Mühlemann O (2013) RNA decay mechanisms: specificity through diversity. Biochim Biophys Acta 1829:487–490
Tomecki R, Drazkowska K, Dziembowski A (2010) Mechanisms of RNA degradation by the eukaryotic exosome. Chembiochem 11:938–945
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 29:2342–2357
Staals RH, Bronkhorst AW, Schilders G et al (2010) Dis3-like 1: a novel exoribonuclease associated with the human exosome. EMBO J 29:2358–2367
Lykke-Andersen S, Tomecki R, Jensen TH et al (2011) The eukaryotic RNA exosome: same scaffold but variable catalytic subunits. RNA Biol 8:61–66
Bonneau F, Basquin J, Ebert J et al (2009) The yeast exosome functions as a macromolecular cage to channel RNA substrates for degradation. Cell 139:547–559
Malet H, Topf M, Clare DK et al (2010) RNA channelling by the eukaryotic exosome. EMBO Rep 11:936–942
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 48:133–144
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 41:3845–3858
Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127:1223–1237
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 14:15–22
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 29:717–728
Lebreton A, Tomecki R, Dziembowski A et al (2008) Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature 456:993–996
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 16:56–62
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 37:1127–1140
Nagarajan VK, Jones CI, Newbury SF et al (2013) XRN 5′→3′ exoribonucleases: structure, mechanisms and functions. Biochim Biophys Acta 1829:590–603
Jinek M, Coyle SM, Doudna JA (2011) Coupled 5′ nucleotide recognition and processivity in Xrn1-mediated mRNA decay. Mol Cell 41:600–608
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 19:1324–1331
Pellegrini O, Mathy N, Condon C et al (2008) In vitro assays of 5′ to 3′-exoribonuclease activity. Methods Enzymol 448:167–183
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 42:1270–1290
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 285:17574–17583
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
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.
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Tomecki, R., Drazkowska, K., Krawczyk, A., Kowalska, K., Dziembowski, A. (2015). Purification of Eukaryotic Exoribonucleases Following Heterologous Expression in Bacteria and Analysis of Their Biochemical Properties by In Vitro Enzymatic Assays. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_25
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DOI: https://doi.org/10.1007/978-1-4939-2214-7_25
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