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High-throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3'-UTRs

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

Reporting period: 2018-09-01 to 2020-02-29

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.
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 mechanisms that are necessary for controlling bacterial adaptability and development.

3’UTRs generated by transcriptional terminators read-through events:
In addition to bona fide 3’UTRs, transcriptional termination read-through events (TRTE) could also generate longer 3’UTRs that affect expression of genes encoded in the opposite DNA strand. Based on our previous RNA-sequencing data, we estimated that 30% of the overlapping transcription in S. aureus originates from the read-through of transcriptional terminators (TTs). To further understand this mechanism and the impact of antisense-RNAs (asRNA) in gene expression, we have focused on studying the example of the asRNA that affects lexA, the gene encoding the SOS-response regulator. The lexA-asRNA was generated by an unknown TRTE occurring on the TT of sbrB. Interestingly, the PsbrB promoter requires the alternative sigma factor B (SigB) to initiate transcription, which is activated when bacteria faces stress. In order to demonstrate that the asRNA had an impact on the SOS response, we constructed strains that either lacked the sbrB promoter or carried a constitutive one in its place. Such strains were challenged with Mitomycin C (MMC), an antibiotic that activates the SOS response. Results showed that when the asRNA was constitutively expressed, bacteria were more resistant to MMC action. Such effect was consistent with an increased activity of the RecA promoter, which is affected when the SOS response is derepressed by LexA. This observation indicated a functional consequence on the expression of the genes because of TRTEs. In particular, we showed the first ever described TRTE capable of generating a functional asRNA that links the cellular stress and the SOS response. This finding is highly relevant for understanding the effect of pervasive transcription, present not only in S. aureus genome but also in almost every bacterium.

In summary, we have showed how most of the RBPs analysed are simultaneously expressed in S. aureus, suggesting a very complex regulatory network. The characterization of the first RBP targetome map of this pathogen indicated that CspA could positively and negatively regulate the expression of its direct targets. Detailed target analysis allowed us to identify the autoregulation molecular mechanisms of CspA (Caballero et al, NAR, 2018). Besides, we identified new bacterial 3’UTRs with regulatory capacities, one of them controlling S. aureus genes associated to virulence and also found that a transcriptional read-though event generates an antisense RNA connecting the cellular stress with the SOS response pathway. All these new post-transcriptional regulatory mechanisms will significantly push forward our knowledge of bacterial gene expression control.
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.