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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9(8):578–589
Sauer E (2013) Structure and RNA-binding properties of the bacterial LSm protein Hfq. RNA Biol 10(4):610–618
Wagner EG (2013) Cycling of RNAs on Hfq. RNA Biol 10(4):619–626
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):294–298
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):19292–19297
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):3546–3556
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):13059–13064
Sauer E, Weichenrieder O (2011) Structural basis for RNA 3′-end recognition by Hfq. Proc Natl Acad Sci U S A 108(32):13065–13070
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):9396–9401
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):e6213
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):4239–4245
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):e1000163
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):266–275
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Nature Singapore Pte Ltd.
About this protocol
Cite this protocol
Han, Y. (2018). Purification of Hfq-Associated RNAs with RNA Immunoprecipitation (RIP). In: Yang, R. (eds) Yersinia Pestis Protocols. Springer Protocols Handbooks. Springer, Singapore. https://doi.org/10.1007/978-981-10-7947-4_10
Download citation
DOI: https://doi.org/10.1007/978-981-10-7947-4_10
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-10-7946-7
Online ISBN: 978-981-10-7947-4
eBook Packages: Springer Protocols