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

EMBO J. 2017 Feb 1;36(3):374-387. doi: 10.15252/embj.201694639. Epub 2016 Nov 11.

Abstract

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.

Keywords: CRAC; EHEC; CLIP‐Seq; enterohaemorrhagic E. coli; non‐coding RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endoribonucleases / metabolism*
  • Escherichia coli / chemistry*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial*
  • Protein Binding
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • RNA, Small Untranslated / analysis*
  • RNA, Small Untranslated / genetics
  • RNA, Small Untranslated / isolation & purification
  • Sequence Analysis, DNA

Substances

  • RNA, Messenger
  • RNA, Small Untranslated
  • Endoribonucleases
  • ribonuclease E