An in vivo map of Hfq binding sites in sRNAs and target mRNAs

Hfq is a nucleic acid binding protein with manifold roles in the control of bacterial gene expression. Recognised predominantly as a molecular platform, Hfq facilitates the association of small RNAs with target mRNAs to regulate translation and turnover. The protein has been known to have strong preferences for adenylate- and uridylate-rich RNA, and several general binding motifs such as A-R-N, A-R-N-N, or A-A-Y-Y-A-A-Y-A-A have been proposed based on in vitro work (1, 2). However, precisely where Hfq binds sRNAs and target mRNAs in vivo is not understood.

We have begun a systematic study in Salmonella to generate a high-resolution in vivo interaction map of Hfq binding sites on cellular RNAs, similar to recent studies of eukaryotic RNA-binding proteins (3, 4). To this end, Hfq-RNA complexes are subjected to UV cross-linking in vivo, followed by denaturing purification of Hfq from cellular extracts, and partial ribonucleolytic digestion of the co-purified RNA, which is then analyzed by Solexa deep sequencing. Using this technique, we hope to answer questions pertaining to binding motifs of Hfq in sRNAs and messengers, localization of the targeted RNA molecules, patterns of RNA competition for Hfq binding, and kinetics of Hfq recruitment during stress responses.