A method for in vivo identification of bacterial small RNA ‐binding proteins

Small bacterial regulatory RNAs ( sRNA s) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA‐binding protein. However, not all sRNA s rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNA s. Here, we describe a methodology for identifying in vivo ‐binding proteins of sRNA s, developed using the iron‐responsive PrrF and PrrH sRNA s of Pseudomonas aeruginosa . RNA was isolated from iron‐depleted cultures, which were irradiated to cross‐link nucleoprotein complexes. Subsequently, PrrF‐ and PrrH‐protein complexes were enriched using cDNA “bait”, and enriched RNA‐protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF‐ and PrrH‐binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA “bait” as compared to the PrrH cDNA “bait”, suggesting Hfq has a stronger binding affinity for the PrrF sRNA s in vivo . Hfq binding to the PrrF and PrrH sRNA s was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa . As such, this study demonstrates that in vivo cross‐linking coupled with sequence‐specific affinity chromatography and tandem mass spectrometry (SSAC‐MS/MS) is an effective methodology for unbiased identification of bacterial sRNA ‐binding proteins.