Small RNA interactome of pathogenic E. coli revealed through crosslinking of RN ase E

RNA sequencing studies have identified hundreds of non‐coding RNA s in bacteria, including regulatory small RNA ( sRNA ). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA – RNA interactions in bacteria. Here we demonstrate that in vivo sRNA – mRNA duplexes can be recovered using UV ‐crosslinking, ligation and sequencing of hybrids ( CLASH ). Many sRNA s recruit the endoribonuclease, RN ase E, to facilitate processing of mRNA s. We were able to recover base‐paired sRNA – mRNA duplexes in association with RN ase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA – mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA – mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNA s. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli . Numerous sRNA interactions were also identified with non‐coding RNA s, including sRNA s and tRNA s, demonstrating the high complexity of the sRNA interactome.