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Computational Analysis, Biochemical Purification, and Detection of tRNA-Derived Small RNA Fragments

  • Simon P. Keam
  • Andrew Sobala
  • David T. Humphreys
  • Catherine M. Suter
  • Gyorgy HutvagnerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)

Abstract

The rapidly growing list of small RNA species generated by next-generation sequencing technologies has accelerated the development of new bioinformatics tools for their detection. Small RNAs generated from tRNAs, transfer RNA-derived fragments (tRFs), represent a novel challenge in accurately identifying and distinguishing them from random degradation products of tRNAs. Here, we describe a bioinformatics approach to detect tRFs in next-generation sequencing libraries. We also present a biochemical purification protocol for enriching 5′ tRFs and separating them from miRNAs. And finally, we suggest reliable methods for detecting and quantifying tRFs.

Keywords

Transfer RNA fragments tRF Deep sequencing Bioinformatics Ionic exchange chromatography Northern blotting Quantitative PCR 

Notes

Acknowledgements

This work was supported by the ARC DP 130103027. G.H. is an ARC Future Fellow.

References

  1. 1.
    Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6):1077–1084PubMedCrossRefGoogle Scholar
  2. 2.
    Cole C et al (2009) Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 15(12):2147–2160PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Lee YS et al (2009) A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 23(22):2639–2649PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Ivanov P et al (2011) Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 43(4):613–623PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Sobala A, Hutvagner G (2013) Small RNAs derived from the 5′ end of tRNA can inhibit protein translation in human cells. RNA Biol 10(4):553–563PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Gebetsberger J et al (2012) tRNA-derived fragments target the ribosome and function as regulatory non-coding RNA in Haloferax volcanii. Archaea 2012:260909PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Haussecker D et al (2010) Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16(4):673–695PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Wang Q et al (2013) Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Mol Ther 21(2):368–379PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Kent WJ et al (2002) The human genome browser at UCSC. Genome Res 12(6):996–1006PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Chan PP, Lowe TM (2009) GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res 37(Database issue):D93–D97PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Schattner P, Brooks AN, Lowe TM (2005) The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33(Web Server issue):W686–W689PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39(Database issue):D152–D157PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Sobala A, Hutvagner G (2011) Transfer RNA-derived fragments: origins, processing, and functions. Wiley Interdiscip Rev RNA 2(6):853–862PubMedCrossRefGoogle Scholar
  14. 14.
    Eddy SR, Durbin R (1994) RNA sequence analysis using covariance models. Nucleic Acids Res 22(11):2079–2088PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Chen C et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Simon P. Keam
    • 1
  • Andrew Sobala
    • 2
  • David T. Humphreys
    • 3
  • Catherine M. Suter
    • 3
  • Gyorgy Hutvagner
    • 1
    Email author
  1. 1.Faculty of Engineering and Information Technology, Centre of Health TechnologiesUniversity of Technology, SydneySydneyAustralia
  2. 2.Department of Infectious DiseasesKing’s College London School of MedicineLondonUK
  3. 3.Molecular, Structural and Computational Biology DivisionVictor Chang Cardiac Research InstituteDarlinghurstAustralia

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