Periodic Reporting for period 4 - BactRNA (Bacterial small RNAs networks unravelling novel features of transcription and translation)
Reporting period: 2024-03-01 to 2025-08-31
Regulation of gene expression plays a key role in the ability of bacteria to rapidly adapt to changing environments and to colonize extremely diverse habitats. It has been known for decades that multiple regulators, among which two-component systems (TCSs), control the transcription of many bacterial genes. More recently, the discovery of a plethora of small regulatory RNAs (sRNAs) and their characterization highlighted the importance of post-transcriptional regulation in bacterial gene expression. It is now clear that the timely expression of many bacterial genes responds to a complex network of both transcriptional and post-transcriptional regulators. However, the properties of the resulting mixed regulatory circuits on the dynamics of gene expression and in the bacterial adaptive response have been poorly addressed so far.
A first major objective of the BactRNA project was to tackle this question by characterizing the circuits that are formed between two widespread classes of bacterial regulators, the sRNAs and the two-component systems, and deciphering their importance in bacterial physiology and virulence. In this regard, results obtained in the course of the BactRNA project not only identified multiple examples of sRNAs controlling TCS synthesis, resulting in a possible connection between different TCSs. They also unraveled the ability of specific antisense RNAs, whose regulatory action is typically restricted to their cognate sense gene, to control expression of multiple genes in trans. Furthermore, the detailed study of one mixed circuit involved in bacterial virulence revealed an unexpected example of a dual feedback control of a TCS mediated either by a small protein (negative feedback) or by the upstream gene in the same operon (positive feedback).
In addition, the study of sRNAs also led to major breakthroughs regarding the basic mechanisms of gene expression, and especially the crucial step of translation initiation. For instance, we previously showed that repressor sRNAs can target activating stem-loop structures located within the coding region of mRNAs that promote translation initiation, in striking contrast with the previously recognized inhibitory role of mRNA structures in translation.
A second major objective of BactRNA was thus to address how these secondary structures or more generally other mRNA elements impact translation, and their possible relation with sRNA control. For this, we studied the action of sRNAs on mRNAs known or suspected to carry translation activating stem-loops, which highlighted exquisite details of sRNA function, among which the role of sRNA transcriptional terminators in gene regulation, or the control of a single gene subject to both sRNA- and riboswitch-control. Most importantly, we also performed a transcriptome-wide analysis of the contacts between the 30S ribosomal small subunit and the mRNAs, which showed multiple binding-sites outside of the translation initiation region, thereby revisiting the canonical model of 30S recruitment and translation initiation in bacteria.
Overall, by identifying new actors and new modes of regulation of bacterial gene expression, this fundamental research project greatly improved our understanding of how these microorganisms can so rapidly and successfully adapt to many different environments. On the longer term, results of this project may provide clues towards the development of anti-bacterial strategies, which is essential given the current antibioresistance threat.
A first major objective of the BactRNA project was to tackle this question by characterizing the circuits that are formed between two widespread classes of bacterial regulators, the sRNAs and the two-component systems, and deciphering their importance in bacterial physiology and virulence. In this regard, results obtained in the course of the BactRNA project not only identified multiple examples of sRNAs controlling TCS synthesis, resulting in a possible connection between different TCSs. They also unraveled the ability of specific antisense RNAs, whose regulatory action is typically restricted to their cognate sense gene, to control expression of multiple genes in trans. Furthermore, the detailed study of one mixed circuit involved in bacterial virulence revealed an unexpected example of a dual feedback control of a TCS mediated either by a small protein (negative feedback) or by the upstream gene in the same operon (positive feedback).
In addition, the study of sRNAs also led to major breakthroughs regarding the basic mechanisms of gene expression, and especially the crucial step of translation initiation. For instance, we previously showed that repressor sRNAs can target activating stem-loop structures located within the coding region of mRNAs that promote translation initiation, in striking contrast with the previously recognized inhibitory role of mRNA structures in translation.
A second major objective of BactRNA was thus to address how these secondary structures or more generally other mRNA elements impact translation, and their possible relation with sRNA control. For this, we studied the action of sRNAs on mRNAs known or suspected to carry translation activating stem-loops, which highlighted exquisite details of sRNA function, among which the role of sRNA transcriptional terminators in gene regulation, or the control of a single gene subject to both sRNA- and riboswitch-control. Most importantly, we also performed a transcriptome-wide analysis of the contacts between the 30S ribosomal small subunit and the mRNAs, which showed multiple binding-sites outside of the translation initiation region, thereby revisiting the canonical model of 30S recruitment and translation initiation in bacteria.
Overall, by identifying new actors and new modes of regulation of bacterial gene expression, this fundamental research project greatly improved our understanding of how these microorganisms can so rapidly and successfully adapt to many different environments. On the longer term, results of this project may provide clues towards the development of anti-bacterial strategies, which is essential given the current antibioresistance threat.
Overall, our work highlighted the sophistication and the diversity of the mechanisms of bacterial gene regulation. This includes new details of sRNA action, such as the functional redundancy between two different isoforms of a single sRNA controlling the same target via different mechanisms, the dual riboswitch- and sRNA-mediated control of a single mRNA, as well as the demonstration of post-transcriptional control by sRNAs transcriptional terminators. The connections between the transcriptional and post-transcriptional regulatory networks were also further studied and unraveled novel regulatory circuits relying on two-component systems important for virulence, sRNA and small proteins; they also led to the unexpected demonstration that some antisense RNAs can also act via imperfect base-pairing on multiple trans-encoded targets. Finally, we addressed the translational control of mRNAs, first by identifying new translation activating stem-loops or elements that could be important for their action and, second, by mapping binding-sites of 30S or 70S ribosomes transcriptome-wide. This last analysis revealed hundreds of 30S binding-sites outside of the translation initiation regions, that, at least for these that were characterized in more detail, are essential for optimal translation.
As for the exploitation and dissemination of these results, we have published six papers in relation with the BactRNA project (4 research articles, 1 invited review article, and 1 invited commentary, see section Publications for details). Another research article is available as a preprint on BioRxiv (https://doi.org/10.1101/2025.04.04.647229(opens in new window)) and was submitted to Molecular Cell in November 2025. Three other papers are in preparation and close to be submitted. They are signed as first author by Jade Mathis de Fromont (submission planned in December 2025), Alexey Korepanov (submission planned in January 2026) or Fanny Quenette (submission planned later in 2026), respectively. In sum, all members of the BactRNA project are co-authors of one or several publications.
The results of BactRNA have also been presented by different members of the project to various meetings and conferences, as well as during invited research seminars. 10 meeting presentations were funded by BactRNA (M1 to M10: see Dissemination section), note that the meetings that were canceled or switched to virtual events due to the Covid pandemy are not listed.
Regarding dissemination, results were also communicated to students, either by the members of the BactRNA project who have been involved in teaching, or via the training of students that we regularly host in the lab.
As for the exploitation and dissemination of these results, we have published six papers in relation with the BactRNA project (4 research articles, 1 invited review article, and 1 invited commentary, see section Publications for details). Another research article is available as a preprint on BioRxiv (https://doi.org/10.1101/2025.04.04.647229(opens in new window)) and was submitted to Molecular Cell in November 2025. Three other papers are in preparation and close to be submitted. They are signed as first author by Jade Mathis de Fromont (submission planned in December 2025), Alexey Korepanov (submission planned in January 2026) or Fanny Quenette (submission planned later in 2026), respectively. In sum, all members of the BactRNA project are co-authors of one or several publications.
The results of BactRNA have also been presented by different members of the project to various meetings and conferences, as well as during invited research seminars. 10 meeting presentations were funded by BactRNA (M1 to M10: see Dissemination section), note that the meetings that were canceled or switched to virtual events due to the Covid pandemy are not listed.
Regarding dissemination, results were also communicated to students, either by the members of the BactRNA project who have been involved in teaching, or via the training of students that we regularly host in the lab.
This project unraveled new insight in gene expression and gene regulation in bacteria. This ranges from newly characterized regulatory circuits relying on small RNAs and two-component systems (and possibly small proteins) to the identification of new modes of action of sRNAs. Another major finding of this project is that hundreds of binding-sites for the 30S small ribosomal subunit are present in mRNA leaders outside of the translation initiation regions, thereby challenging the current model of the key step of translation initiation in bacteria.
Importantly, some of these findings relied on the development of new methodologies during this project, such as the “EASY-edit” genetic editing tool or the “30S-seq” method, and that will find many future applications.
Importantly, some of these findings relied on the development of new methodologies during this project, such as the “EASY-edit” genetic editing tool or the “30S-seq” method, and that will find many future applications.