Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ReguloBac-3UTR (High-throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3\'-UTRs)

Teaser

Knowing how bacteria control gene expression is crucial for a better understanding and managing of infection diseases, antimicrobial resistance and/or biotechnological processes. Although gene regulation based on non-coding RNAs has been extensively studied during last years...

Summary

Knowing how bacteria control gene expression is crucial for a better understanding and managing of infection diseases, antimicrobial resistance and/or biotechnological processes. Although gene regulation based on non-coding RNAs has been extensively studied during last years, the potential of the 3’ untranslated regions (3’UTRs) of messenger RNAs (mRNAs) as putative regulatory elements has been disregarded in bacteria. In contrast, eukaryotic 3’UTRs are widely considered key post-transcriptional regulatory elements, controlling development and behaviour of organisms. The aim of the ReguloBac-3UTR project is to identify and characterize the 3’UTR-medianted regulatory mechanisms and its associated RNA-binding proteins to demonstrate that the bacterial 3’UTRs are as relevant as their eukaryotic counterparts. Gaining knowledge of this respect should significantly improve our capacities in manipulating gene expression and dealing with bacterial associated problems.

Work performed

Since regulation of gene expression by 3’UTRs might occur through different mechanisms, the ReguloBac-3UTR project was designed to confront the problem from different angles:

Identification and characterization of bona fide 3’UTRs:
In order to identify bona fide regulatory 3’UTRs, we created a ranked list of putative functional 3’UTRs based on our previous transcriptomic data. For this purpose, we considered the length of the 3’UTR, the functional relevance of the protein encoded in the mRNA and the presence of antisense and small RNAs in the region of interest. The top-listed genes were tagged to analyse if their expression was affected in vivo when lacking the 3’UTR. We found that the deleting the 3’UTRs altered the amount of protein production for some mRNAs, supporting the idea that 3’UTR mediated-regulation is also present in prokaryotes. To analyse the functional relevance of such regulation, we focused on the 3’UTR of an mRNA encoding a transcriptional regulator that controls S. aureus virulence. By generating chromosomal 3’UTR mutants, we found that S. aureus lost its haemolytic capacities. These results showed that a 3’UTR is required to control essential virulence-related genes in S. aureus, one of the most important pathogens worldwide. We are currently determining the molecular mechanism of this regulation.

Identification of RNA-binding proteins associated to 3’UTR-mediated regulation:
In eukaryotes, it has been shown that numerous RNA binding proteins (RBPs) participate in 3’UTR-mediated gene expression control. Therefore, it is expected of bacterial RBPs to have similar functions. We reasoned that elucidating the targetome maps of representative protein members of the different S. aureus RBP families would allow us to identify some of them. First, we chromosomally tagged the chosen RBP examples and analysed their expression pattern along the growth curve. Interestingly, most of them were expressed at the same time suggesting a very complex regulatory network. To determine their targetomes, we initially focussed on those RBPs with described orthologue functions in eukaryotes. For example, the Unr protein, which participates in 3’UTR-mediated regulation in eukaryotes and contains similar domains to those found in the cold-shock protein (CSP) family in bacteria. CSPs are RNA chaperones for which most of their targets remain unknown. S. aureus genome encodes three CSPs (CspA, CspB and CspC). The first of them, CspA, is amongst the most abundantly expressed proteins in this pathogen. Combining targetomic and proteomic data, we determined the CspA regulon and showed how this RBP can modulate positively and negatively the expression of its targets. Although a positive regulation from CspA was expected due to its putative RNA melting activity, which would facilitate ribosome progression, a negative regulation indicated that CspA could have additional functions to those previously anticipated. Therefore, we aimed to further study the molecular mechanisms behind such negative regulation. We chose the cspA mRNA as an example. CspA targeted its own mRNA and, as a result, repressed its protein expression. We discovered that a U-rich motif located at a stem-loop within the cspA 5’UTR was important for transcript recognition. Such secondary structure had been previously described as a target of the double-stranded endoribonuclease RNase III. We revealed that the mRNA cleavage by RNase III lead to an improved translation in vivo and that CspA antagonized with RNase III activity by disrupting the stem loop. This finding portrayed CspA as a putative RNase III-antagonist, which could apply to other RNase III targets (Caballero et al, 2018). In addition, this highlights the importance of regulatory RNA elements, within the mRNAs, and how RBPs can alter translation by modifying them. We believe that determining other RBP targetome maps (currently in course) will help uncovering new regulatory mechani

Final results

The discovery of new 3’UTR-mediated regulatory mechanisms in bacteria will help identifying new potential targets for the development of novel antimicrobials. This would contribute in dealing with the challenging multi-drug antibiotic resistance, a worldwide problem. In addition, demonstrating that 3’UTRs constitute a new layer of regulation in bacteria, will help improving our basic knowledge of microbiology with significant repercussions on other fields such as synthetic biology and biotechnology.

Website & more info

More info: http://idab.es/grupo-de-investigacion-de-regulacion-genica-bacteriana/erc-cog-regulobac-3-utrs/.