High-Throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3'-utrs
In eukaryotes, untranslated regions located at the 3′ end (3’UTRs) of messenger RNAs (mRNAs) have been proved to be key post-transcriptional regulatory elements controlling almost every single biological process. In contrast, in bacteria, most studies regarding post-transcriptional regulation have been mainly focused on specific non-coding RNAs and 5’UTRs, which often carry riboswitches or thermosensors. Remarkably, bacterial 3’UTRs have been largely disregarded and have not been considered as potential regulators. Recently, we found that a 3’UTR modulates biofilm formation in S. aureus through its interaction with the 5’UTR encoded in the same mRNA. This mechanism resembles eukaryotic mRNA circularization. Also, a 3’UTR that contributes to cellular homeostasis by promoting hilD mRNA turnover was recently shown in Salmonella. Although both studies are pioneering showing the potential of bacterial 3’UTRs as regulatory elements, many questions still remain to be answered. Are 3’UTRs roles conserved in bacterial species? Do 3’UTRs contain specific regulatory sequences or secondary RNA structures? Are transcriptional terminator sequences relevant for certain 3’UTRs? Are 3’UTRs specifically recognized by RNA-binding proteins? Might 3’UTRs be responsible for bacterial speciation? Might bacterial 3’UTRs be the ancestors of eukaryotic 3’UTR evolution? To achieve these questions, here we propose a high-throughput analysis based on the development of specialized dual-reporter libraries to identify in vivo functional 3’UTRs by fluorescence-activated cell sorting coupled to RNA sequencing. Also the pool of RNA-binding proteins associated to 3’UTRs will be identified by global MS2-tagging and mass spectrometry. Examples of 3’UTRs belonging to physiologically important genes will be selected to deeply study regulatory mechanisms at the molecular and single cell levels. We expect that this project will largely change the view of post-transcriptional regulation in bacteria.