Periodic Reporting for period 4 - ReguloBac-3UTR (High-throughput in vivo studies on posttranscriptional regulatory mechanisms mediated by bacterial 3'-UTRs)
Berichtszeitraum: 2020-03-01 bis 2020-08-31
Knowing how bacteria control gene expression is crucial for a better understanding and managing of infection diseases, antimicrobial resistance and/or biotechnological processes. Although regulation based on non-coding RNAs has been extensively studied during the past years, the potential of the 3’ untranslated regions (3’UTRs) from messenger RNAs (mRNAs) and RNA-binding proteins (RBPs) as putative regulatory elements had not been considered until more recent times. In the ReguloBac-3UTR project we identified and characterized regulatory 3’UTRs in bacteria including Staphylococcus aureus, a life-threatening pathogen used as model in these studies. Additionally, we demonstrated their evolutionary selection and impact in creating species-specific post-transcriptional regulatory networks. We also described the functional specificity and the post-transcriptional regulation of the cold shock proteins, a family of widely distributed RNA-binding proteins that are crucial for stress adaptation. From our perspective, these findings shall improve our capacities in manipulating gene expression and dealing with bacterial associated problems.
In order to identify bacterial 3’UTRs with regulatory capacities, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins among closely-related bacterial species of the Staphylococcus genus. Unexpectedly, we found that most of the 3′UTRs presented a high sequence diversity. RNA-seq experiments revealed that their 3’UTRs were also different in length, suggesting a selection of species-specific functional 3’UTRs throughout evolution (P. Menendez-Gil et al, NAR, 2020). We showed that sequence variations in the 3’UTRs might occur through different processes, including gene rearrangements, local nucleotide changes and transposition of insertion sequences. We further analysed the functionality of the icaR, ftnA and rpiRc 3’UTRs from Staphylococcus aureus, one of the most clinically relevant pathogens worldwide. We found that these 3’UTRs modulate relevant physiological processes of S. aureus, including biofilm formation, iron homeostasis and virulence. Swapping of the 3’UTRs of the orthologous genes from several staphylococcal species revealed that the nucleotide variations in the 3’UTR sequences altered the mRNA and protein levels. This indicated that the 3’UTRs may have distinct functional roles depending of the bacterial species. We confirmed that the 3’UTR variability is a widespread phenomenon in bacteria (P. Menendez-Gil et al, NAR, 2020; FiMB, 2021). We also found that even single-nucleotide polymorphisms (SNPs) in transcriptional terminators (TT) from different S. aureus strains might affect the expression of neighbouring genes. The SNPs lead to a malfunctioning of the TTs, which causes transcriptional read-through events that generate long 3’UTRs overlapping with their neighbouring genes (Bastet et al, manuscript in preparation). These findings indicated that gene organizations across the chromosomes should also be considered when studying the expression of a particular gene. On this subject, we collaborated with Prof. Iñigo Lasa to describe a novel transcriptional organization in bacteria that coordinates gene expression among adjacent genes (Saenz-Lahoya et al, PNAS, 2019; Toledo-Arana & Lasa, Mol. Microbiol, 2020).
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.
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.
The discovery of novel post-transcriptional regulatory mechanisms in bacteria will help identifying new potential targets for the development of novel antimicrobials and/or anti-virulence drugs. This would contribute in dealing with the challenging multi-drug antibiotic resistance, a worldwide problem. In addition, by demonstrating that bacteria could control gene expression using species specific 3’UTR-mediated regulatory mechanisms, we provided options to develop drugs targeting a specific pathogenic bacterial species. This could help with the fighting against specific pathogens without affecting the normal microbiota. For that reason, we believe that we have gone a step further in increasing our basic knowledge of microbiology that may translate into significant advancements on other fields such as medicine, synthetic biology and biotechnology.