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.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
Shafagh A
SA
Waters
The Roslin Institute, University of Edinburgh, Edinburgh, UK.
Sean P
SP
McAteer
MRC Human Genetic Unit, University of Edinburgh, Edinburgh, UK.
Grzegorz
0000-0002-7924-2744
G
Kudla
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.|Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
Ignatius
I
Pang
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.|Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
Nandan P
NP
Deshpande
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
Timothy G
TG
Amos
Peter Doherty Institute, University of Melbourne, Melbourne, Victoria, Australia.
Kai Wen
KW
Leong
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.|Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
Marc R
MR
Wilkins
Peter Doherty Institute, University of Melbourne, Melbourne, Victoria, Australia.
Richard
R
Strugnell
The Roslin Institute, University of Edinburgh, Edinburgh, UK.
David L
DL
Gally
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK j.tree@unsw.edu.au d.tollervey@ed.ac.uk.
David
0000-0003-2894-2772
D
Tollervey
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia j.tree@unsw.edu.au d.tollervey@ed.ac.uk.
Jai J
0000-0001-8233-0975
JJ
Tree
D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D058727:RNA, Small Untranslated / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D012333:RNA, Messenger; D058727:RNA, Small Untranslated; D004722:Endoribonucleases; C022537:ribonuclease E

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