Purification of Hfq-Associated RNAs with RNA Immunoprecipitation (RIP)
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 wordsYersinia pestis Hfq sRNA RNA immunoprecipitation (RIP)