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Tips and Tricks to Probe the RNA-Degrading Activities of Hyperthermophilic Archaeal β-CASP Ribonucleases

  • Duy Khanh Phung
  • Béatrice Clouet-d’OrvalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

The importance of ribonucleases in posttranscriptional control of gene expression has been established in Eukarya and Bacteria for over a decade. However, this process has been overlooked in Archaea, which are of universal importance to elucidate fundamental biological mechanisms and to study the evolution of life on Earth. Very few ribonucleolytic activities have been reported in Archaea, and RNA metabolism pathways wait to be described. Recently we have identified two major groups of archaeal ribonucleases, aCPSF1 and aRNase J, which are members of the β-CASP metallo-β-lactamase family. Here, we describe in vitro methods to characterize the endo- and exoribonucleolytic activities of hyperthermophilic archaeal β-CASP ribonucleases. The use of various labeled RNA substrates allows defining the specificity of RNA cleavage and the directionality of the exoribonucleolytic trimming activity of the archaeal enzymes which work at high temperature. Elucidating in vitro ribonucleolytic activities is one step toward the understanding of the role of β-CASP ribonucleases in RNA metabolism pathways in archaeal cells.

Key words

β-CASP ribonucleases Exoribonucleolytic activity Endoribonucleolytic activity Archaea Thermococcale Hyperthermophilic enzymes 

Notes

Acknowledgments

This work is supported by the Centre National de la Recherche Scientique (CNRS) with additional funding from the Agence Nationale de la Recherche (ANR) [BLAN08-1_329396] and from the Université de Toulouse (UPS). D.K.P. is supported by a Ph.D. scholarship from the French “Ministère de l’Éducation nationale, de l’Enseignement supérieur et de la Recherche.”

References

  1. 1.
    Stoecklin G, Muhlemann O (2013) RNA decay mechanisms: specificity through diversity. Biochim Biophys Acta 1829:487–490PubMedCrossRefGoogle Scholar
  2. 2.
    Evguenieva-Hackenberg E, Klug G (2009) RNA degradation in Archaea and Gram-negative bacteria different from Escherichia coli. Prog Mol Biol Transl Sci 85:275–317PubMedCrossRefGoogle Scholar
  3. 3.
    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
  4. 4.
    Phung DK, Rinaldi D, Langendijk-Genevaux PS et al (2013) Archaeal beta-CASP ribonucleases of the aCPSF1 family are orthologs of the eukaryal CPSF-73 factor. Nucleic Acids Res 41:1091–1103PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Hasenohrl D, Konrat R, Blasi U (2011) Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5′-to-3′ directional decay and 5′-end protection of mRNA in Crenarchaeota. RNA 17:99–107PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Martens B, Amman F, Manoharadas S et al (2013) Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2. PLoS One 8:e76569PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Callebaut I, Moshous D, Mornon JP et al (2002) Metallo-beta-lactamase fold within nucleic acids processing enzymes: the beta-CASP family. Nucleic Acids Res 30:3592–3601PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Dominski Z (2007) Nucleases of the metallo-beta-lactamase family and their role in DNA and RNA metabolism. Crit Rev Biochem Mol Biol 42:67–93PubMedCrossRefGoogle Scholar
  9. 9.
    Condon C, Gilet L (2011) The metallo-β-lactamase Family of Ribonucleases, vol 26, Nucleic Acids and Molecular Biology. Springer, Berlin Heidelberg, pp 245–267Google Scholar
  10. 10.
    Dominski Z, Carpousis AJ, Clouet-d’Orval B (2013) Emergence of the beta-CASP ribonucleases: highly conserved and ubiquitous metallo-enzymes involved in messenger RNA maturation and degradation. Biochim Biophys Acta 1829:532–551PubMedCrossRefGoogle Scholar
  11. 11.
    Li de la Sierra-Gallay I, Zig L, Jamalli A et al (2008) Structural insights into the dual activity of RNase J. Nat Struct Mol Biol 15:206–212PubMedCrossRefGoogle Scholar
  12. 12.
    Mandel CR, Kaneko S, Zhang H et al (2006) Polyadenylation factor CPSF-73 is the pre-mRNA 3′-end-processing endonuclease. Nature 444:953–956PubMedCrossRefGoogle Scholar
  13. 13.
    Even S, Pellegrini O, Zig L et al (2005) Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E. Nucleic Acids Res 33:2141–2152PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Cohen GN, Barbe V, Flament D et al (2003) An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi. Mol Microbiol 47:1495–1512PubMedCrossRefGoogle Scholar
  15. 15.
    Evguenieva-Hackenberg E, Wagner S, Klug G (2008) In vivo and in vitro studies of RNA degrading activities in Archaea. Methods Enzymol 447:381–416PubMedCrossRefGoogle Scholar
  16. 16.
    Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62PubMedCrossRefGoogle Scholar
  17. 17.
    Nolivos S, Carpousis AJ, Clouet-d’Orval B (2005) The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn. Nucleic Acids Res 33:6507–6514PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Phok K, Moisan A, Rinaldi D et al (2011) Identification of CRISPR and riboswitch related RNAs among novel non-coding RNAs of the euryarchaeon Pyrococcus abyssi. BMC Genomics 12:312PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Petrov A, Wu T, Puglisi EV et al (2013) RNA purification by preparative polyacrylamide gel electrophoresis. Methods Enzymol 530:315–330PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Centre National de la Recherche Scientifique, UMR 5100-LMGMCNRS and Université de ToulouseToulouse Cedex 9France
  2. 2.UMR 5100-LMGMCNRS and Université de ToulouseToulouse Cedex 9France

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