RNA interactome capture in E. coli to identify RNA-binding proteins

RNA-binding proteins (RPBs) are deeply involved in 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 poly(A) polymerase I, we drastically increase the fraction of polyadenylated RNA in Escherichia coli, allowing us to pull-down RNA-protein complexes using immobilized oligo-d(T) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding Overall design: Addition of poly(A) tails to E. coli RNA by overexpression of PAPI combined with UV-light crosslinking to allow for purification of polyadenylated RNA along with co.purification of interacting proteins using oligo d(T) beads