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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756737 |
| Date | January 2018 |
| Creators | Iosub, Ira Alexandra |
| Contributors | Granneman, Sander ; Tollervey, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/31477 |
Page generated in 0.0019 seconds