Advertisement

Purification of Hfq-Associated RNAs with RNA Immunoprecipitation (RIP)

  • Yanping Han
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The RNA-binding protein Hfq is considered a central hub of small RNA (sRNA)-mediated regulation and is essential for the bacterial physiology and pathogenesis. It generally forms a homohexameric ring complex containing RNA-binding sites. Hfq is implicated in the pathogenesis of Yersinia pestis. The identification of Hfq-binding RNA species in vivo is critical for understanding their functions and determining their modes of interaction. The RNA immunoprecipitation (RIP) technique has been used to identify a large number of Hfq-associated RNA species, and can be effectively used to detect Hfq-bound RNAs in vivo, which can be subsequently analyzed with Northern blotting, quantitative PCR, microarray analysis, or deep sequencing. Here, endogenously formed Hfq–RNA complexes of Y. pestis were captured with immunoprecipitation with a monoclonal anti-Flag antibody. The Hfq-associated RNAs were then purified and identified with deep sequencing.

Key words

Yersinia pestis Hfq sRNA RNA immunoprecipitation (RIP) 

References

  1. 1.
    Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9(8):578–589CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sauer E (2013) Structure and RNA-binding properties of the bacterial LSm protein Hfq. RNA Biol 10(4):610–618CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wagner EG (2013) Cycling of RNAs on Hfq. RNA Biol 10(4):619–626CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Morita T, Aiba H (2011) RNase E action at a distance: degradation of target mRNAs mediated by an Hfq-binding small RNA in bacteria. Genes Dev 25(4):294–298CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Link TM, Valentin-Hansen P, Brennan RG (2009) Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. Proc Natl Acad Sci U S A 106(46):19292–19297CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Schumacher MA, Pearson RF, Moller T, Valentin-Hansen P, Brennan RG (2002) Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J 21(13):3546–3556CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Otaka H, Ishikawa H, Morita T, Aiba H (2011) PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action. Proc Natl Acad Sci U S A 108(32):13059–13064CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Sauer E, Weichenrieder O (2011) Structural basis for RNA 3′-end recognition by Hfq. Proc Natl Acad Sci U S A 108(32):13065–13070CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sauer E, Schmidt S, Weichenrieder O (2012) Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition. Proc Natl Acad Sci U S A 109(24):9396–9401CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Geng J, Song Y, Yang L, Feng Y, Qiu Y, Li G, Guo J, Bi Y, Qu Y, Wang W et al (2009) Involvement of the post-transcriptional regulator Hfq in Yersinia pestis virulence. PLoS One 4(7):e6213CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bai G, Golubov A, Smith EA, McDonough KA (2010) The importance of the small RNA chaperone Hfq for growth of epidemic Yersinia pestis, but not Yersinia pseudotuberculosis, with implications for plague biology. J Bacteriol 192(16):4239–4245CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sittka A, Lucchini S, Papenfort K, Sharma CM, Rolle K, Binnewies TT, Hinton JC, Vogel J (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet 4(8):e1000163CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sittka A, Sharma CM, Rolle K, Vogel J (2009) Deep sequencing of salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes. RNA Biol 6(3):266–275CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Yanping Han
    • 1
  1. 1.Beijing Institute of Microbiology and EpidemiologyBeijingChina

Personalised recommendations