RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins

RNA-binding proteins (RPBs) are deeply involved in many fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to globally identifying bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down mRNA-protein complexes. By overexpressing the poly(A) polymerase I, we drastically increase the frequency of polyadenylated RNA in E. coli, allowing us to pull down RNA-protein complexes using immobilized oligo-d(T) as bait. With this approach, we identified 167 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of several proteins previously unknown to interact with RNA, including the uncharacterized protein YhgF. YhgF is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF in vivo RNA targets using CLIP-seq, two of which were verified using electromobility shift assays. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lays the foundation for further studies of the strongly conserved RBP Yhg