Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs 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 sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase 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 mRNAs. 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 RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome. Synopsis A new method uses UV‐crosslinking of RNase E–RNA complexes to map the small RNA–RNA interaction network in bacteria and provide transcriptome‐wide information on small RNA function. The small RNA–RNA interaction network in bacteria is captured by UV‐crosslinking of RNase E–RNA complexes in vivo . RNase E binding sites are identified in a transcriptome‐wide manner. Small RNAs direct target RNA cleavage 3′ of duplexed seed sequences. The enterohaemorrhagic E. coli ‐specific sRNA, Esr41, confers colicin 1A resistance. Graphical Abstract A new method using UV‐crosslinking maps the small RNA–RNA interaction network in bacteria and provides transcriptome‐wide information on small RNA function.