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

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.

Advances and challenges towards the study of RNA-RNA interactions in a transcriptome-wide scale

“…One of the first sequencing-based techniques to decode the RNA interactome of a specific RNA is CLASH (crosslinking, ligation, and sequencing of hybrid) [34][35][36] (Figure 2A). As many RNA interactions are mediated by proteins (e.g., AGO in miRNA-mRNA interaction), UV can be used to crosslink a RRI duplex For biophysical or cellular methods, the experiments have been conducted in many species.…”

“…After bioinformatics analysis of chimeric reads that correspond to RNA-RNA duplexes, the RNA interactions can be identified. CLASH has been applied to characterize different types of RRIs among diverse species, including small RNA centered interactions in bacterial [36], snoRNA-rRNA interactions in yeast [34], and miRNA-mRNA interactions in human [35]. Recently, CLASH was slightly modified to detect RRIs in Escherichia coli [37], in which AMT molecule was also used to crosslink RRIs that are not mediated by proteins.…”

“…For example, the mRNA targets of an enterohemorrhage E. coli (EHEC)-specific sRNA, Esr41, were identified. It is shown that Esr41 can interact with the transcripts of iron transport and storage proteins CirA, ChuA and Bfr, thus influence their expressions [36]. In Ref.…”

“…For instance, both PARIS and SPLASH revealed a big number of mRNA-lncRNA and mRNA-mRNA interactions. Besides, many sRNAmRNA interactions have been revealed by CLASH [36] and a similar technique called RIL-seq [63]. Interaction between sRNA and other kinds of RNAs like ncRNA and tRNA have also been identified but the most enriched ones are sRNA-mRNA interactions [36,63].…”

Cross-Regulation between Bacteria and Phages at a Posttranscriptional Level

“…A recent study applying CLASH (cross-linking, ligation, and sequencing of hybrids) to RNase E revealed multiple direct interaction partners of Esr41 in EHEC (69). Three mRNAs encoding an iron-siderophore complex uptake receptor (cirA) , bacterioferritin (bfr) , and outer membrane heme receptor (chuA) were confirmed to form duplexes with Esr41.…”