Periodic Reporting for period 4 - ReguloBac-3UTR (High-throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3'-UTRs)
Reporting period: 2020-03-01 to 2020-08-31
The ReguloBac-3UTR project was also focused on the characterization of cold shock proteins (CSP), which are RNA chaperones carrying the cold shock domain (CSD). The CSD is widely distributed in all organisms. The number of CSPs is dependent on the bacterial species, ranging from one to eighteen. S. aureus, our model organism, encodes three CSPs paralogs (CspA, CspB and CspC). CspA, is amongst the most abundantly expressed proteins in this pathogen. We demonstrated how S. aureus CSPs are relevant for oxidative and cold stresses adaptation, their functional specificity and post-transcriptional regulation (Caballero et al, 2018, NAR; Catalán-Moreno et al, 2020, Mol Microbiol; Catalán-Moreno et al, 2021, NAR).
First, we determined the CspA regulon and showed that CspA binds hundreds of mRNAs to modulate their expression. Further analyses revealed that CspA is able to repress its own expression by binding to a U-rich motif located at a stem-loop within the cspA 5’UTR, which is targeted by the double-stranded endoribonuclease RNase III. We also showed that the mRNA cleavage by RNase III led to an improved translation in vivo and that CspA antagonized the RNase III activity by disrupting such stem loop. This finding portrayed CspA as a putative RNase III-antagonist, which could apply to other RNase III targets (Caballero et al, NAR, 2018).
Second, we demonstrated that the CspA function could not be restored by CspB nor CspC in a cspA mutant despite the high protein sequence identity between all three CSPs. Detailed molecular analyses unveiled that one evolutionarily selected amino acid variation was sufficient to provide functional specificity among S. aureus CSP paralogs. By creating several chimeric CSPs that interchanged the amino acid differences between CSPs, we found that proline at position 58 of CspA was responsible for the specific control of SigB-dependent phenotypes related to stress adaptation (Catalan-Moreno et al, Mol Microbiol, 2020).
Finally, we unveiled that a thermoregulatory mechanism, which controlled the production of CspB and CspC proteins, was required for S. aureus adaptation when growing at ambient temperatures. We showed that CspB and CspC were post-transcriptionally regulated by complex paralogous RNA structures located in their 5’UTRs, which worked as thermoswitches that induced protein expression at low temperatures. Mutations that fixed the thermoswitches in an OFF configuration meant that the CspB and CspC protein could not be translated and prevented S. aureus growth at 22ºC. This underlined the importance of thermoregulation and their impact in S. aureus survival when away from the host (Catalan-Moreno et al, 2021, NAR).
In summary, this project has contributed to the finding and characterization of several novel post-transcriptional regulatory mechanisms that involve diverse 3’UTRs and RBPs, expanding our knowledge of gene expression control in bacteria. The fact that some of these regulatory mechanisms have been selected through the course of evolution to create diversity among bacterial species increases the relevance of our results.