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ReguloBac-3UTR Report Summary

Project ID: 646869
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - ReguloBac-3UTR (High-throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3'-UTRs)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

Eukaryotic untranslated regions located at the 3′ end (3’UTRs) of messenger RNAs (mRNAs) are key post-transcriptional regulatory elements controlling almost every single biological process. In contrast, post-transcriptional regulation studies in bacteria have been mainly focused on specific non-coding RNAs (both asRNAs and sRNAs) and 5’-UTRs, which often carry riboswitches or thermosensors. However, bacterial 3’UTRs have been largely disregarded as potential regulatory elements. Recently, we found that a long 3’UTR modulates biofilm formation in S. aureus through interaction with the 5’-UTR of its own mRNA. Such mechanism resembles eukaryotic mRNA circularization. In addition, a regulatory long 3’UTR has been also described for Salmonella. This 3’UTR is located at the hilD mRNA to control mRNA turnover and expression of the HilD protein, which is the main regulator of the pathogenicity island SPI1 of Salmonella. Although both studies are pioneers in showing the potential of bacterial 3’UTRs to control important biological processes, multicellular behaviour and virulence respectively, many questions still remain to be answered. Are 3’UTR roles conserved throughout bacterial species? Do 3’UTRs contain specific regulatory sequences and/or secondary RNA structures? Are transcriptional terminators relevant for 3’UTRs? Are 3’UTRs specifically recognized by ribonucleases and/or RNA-binding proteins? Could 3’UTRs be responsible for bacterial speciation? Is bacterial 3’UTR-mediated regulation as general as in eukaryotes? Might bacterial 3’UTRs be the ancestors of eukaryotic 3’UTR evolution?
In order to answer these questions, we propose a high-throughput analysis based on the development of specialized 3’UTRs reporter libraries to identify in vivo genome-wide functional regulatory 3’UTRs by fluorescence-activated cell sorting (FACS) coupled to deep RNA sequencing. In addition, the pool of RNA-binding proteins associated to 3’UTRs will be identified by genome wide approaches. Finally, relevant examples of 3’UTRs belonging to physiologically important genes will be selected to deeply study the regulatory mechanisms at the molecular and single-cell levels. We expect this project to largely change the view of post-transcriptional regulation in bacteria.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Regulation of gene expression through 3’UTRs in bacteria can happen in different ways. For this reason, the project has been designed to confront the problem form different angles.
First, in order to identify putative functional 3’UTRs of the human pathogen Staphylococcus aureus, we created a list of putative functional 3’UTRs considering i) the length of the 3’UTR, ii) the functional relevance of the protein encoded into the mRNA and iii) the presence of antisense and/or small RNAs in the region of interest. The importance of the 3’UTR was tested for the most relevant candidates by labelling them with a 3xFLAG tag and comparing the protein expression of their full mRNA and their 3’UTR deleted versions. To date, we found that deletion of the 3’UTRs affected the expression of at least three mRNAs. In two cases, the differential expression was only observed at the stationary growth phase. This result indicated that an additional factor, a protein or a small RNA, might be interacting with the 3’UTR. Our next goal is to identify which might be these additional factors involved in the 3’UTR-mediated regulation.
Second, to identify genes affected by long 3’UTRs, generated by read-through transcriptional termination events, we performed bioinformatics analysis of RNA-seq transcriptomic data. This analysis revealed that more than two hundred Rho-independent transcriptional terminators (TTs) suffered read-through in S. aureus. Among them, we identified SbrB, a small messenger RNA (mRNA) whose read-through results in a long transcript antisense to lexA gene, the major regulator of SOS response in bacteria. Preliminary results confirmed that the expression of this long antisense transcript was directly controlled by the alternative sigma factor (SigB), the regulator of stress response in bacteria. The generation of a SigB-dependent transcript antisense to lexA might be providing a physical link connecting two of the most important stress response pathways in bacteria. To test this hypothesis, we are currently developing fluorescent reporters that allow monitoring simultaneous expression of the antisense and target gene at a single cell level.
Finally, to identify potential RNA-binding proteins (RBPs) interacting with 3’UTRs, we are performing CLIP assays with selected RBPs. This will allow us to determine the corresponding targetome maps, including the 3’UTRs interactions. For this purpose, we have labelled representative examples of different RBP groups such as RNA helicases and RNA chaperones, including cold shock proteins (CSPs). We found that although all tagged proteins were expressed in the tested conditions, we could only pull down RNA from the CSP group. We thought that the low protein levels that are naturally expressed by the bacteria might be hindering the purification of enough amount of RNA to generate the RNA libraries. To solve this problem, we are currently constructing new strains that will express the RBPs from stronger promoters.
In summary, in this first reporting period we have identified three new bona fide 3’UTRs controlling gene expression in S. aureus. In addition, we found one mRNA suffering SigB-dependent transcriptional read-through and generating a long antisense transcript to lexA (the major SOS response regulator). This example is showing that genes might be organized in clusters connected by long transcripts, providing an additional regulatory layer. Furthermore, we have set up the conditions needed to pull-down the RNAs associated to RNA-binding proteins in S. aureus. If the project continues evolving as expected, we are confident that we will accomplish the proposed objectives.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project is helping to understand the genomic organizations in bacteria that lead to 3’UTR mediated post-transcriptional regulatory mechanisms, which have not been seriously considered yet. This will help improving our basic knowledge in fields such as microbiology, synthetic biology and biotechnology. The finding of new regulatory layers will imply new potential targets for the development of novel antimicrobials that might contribute to deal with the challenging issue of multi-drug antibiotic resistance, a worldwide problem.
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