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

Stenum TS, Kumar AD, Sandbaumhüter FA, Kjellin J, Jerlström-Hultqvist J, Andrén PE, Koskiniemi S, Jansson ET, Holmqvist E

Nucleic Acids Res. 51 (9) 4572-4587 [2023-05-22; online 2023-03-30]

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 direct and global identification of 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 RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Spatial Mass Spectrometry [Collaborative]

PubMed 36987847

DOI 10.1093/nar/gkad216

Crossref 10.1093/nar/gkad216

pmc: PMC10201417
pii: 7092918

Publications 9.5.0