Microorganisms are exposed to constantly changing environments, and consequently
have evolved mechanisms to rapidly adapt their physiology upon stress imposition.
These adaptive responses are coordinated through the rewiring of gene expression
via complex networks that control the transcriptional program and the activity of post-transcriptional
regulators. Although transcription factors primarily determine which
genes are expressed, post-transcriptional regulation has a major role in fine-tuning
the dynamics of gene expression.
Post-transcriptional control is exerted by RNA-binding proteins and small regulatory
RNAs (sRNAs) that bind to mRNA targets and modulate their synthesis, degradation
and translation efficiency. In Escherichia coli, sRNAs associated with an RNA
chaperone, Hfq, are key post-transcriptional regulators, yet the functions of most of
these sRNAs are still unknown. The first step in understanding the roles of sRNAs
in regulating gene expression is to identify their targets. To generate transcriptome-wide
maps of Hfq-mediated sRNA-mRNA binding, we applied CLASH (cross-linking,
ligation and sequencing of hybrids), a method that combines in vivo capture of RNA-RNA
interactions, high-throughput sequencing and computational analyses, in E. coli.
We uncovered thousands of dynamic growth-stage dependent association of Hfq to
sRNAs and mRNAs. The latter confirmed known sRNA-target pairs and identified
additional targets for known sRNAs, as well as novel sRNAs in various genomic
features along with their targets. These data significantly expand our knowledge of
the sRNA-target interaction networks in E.coli. In particular, the Hfq CLASH data
indicated 3’-UTRs of mRNAs as major reservoirs of sRNAs, and the utilization of
these may be more common than anticipated. Our findings also provide mechanistic
insights that ensue from the identification of tens of sRNA-sRNA interactions that
point to extensive sponging activity among regulatory RNAs: many sRNAs appear to
be able to interact and repress the functions of other base-pairing sRNAs. We validated and highlighted the biological significance of some of the CLASH
results by characterizing a 3’-UTR derived sRNA, MdoR (mal-dependent OMP
repressor). This sRNA emerges by processing of the last transcript of malEFG
polycistron, encoding components of maltose transport system. We found MdoR
directly downregulates several major porins, whilst derepressing the maltose-specific
porin LamB via destabilization of its inhibitor, MicA, likely by a sponging mechanism.
Physiologically, MdoR contributes to the remodelling of envelope composition and
links nutrient sensing to envelope stress responses during maltose assimilation.
MdoR is a clear example of how cells integrate circuitry through multiple networks as
part of their adaptive responses and how the CLASH methodology can help expand
our understanding of sRNA-based regulation
