Theme n°1:
RNA based mechanisms of gene expression
Gene expression plays a key role in allowing bacteria to adapt to their environment. While transcription was the first step of gene expression to be shown as subject to regulation, later studies demonstrated that post-transcriptional control exists as well. Furthermore, it has been known for decades that RNA can play a crucial role in regulation of gene expression in bacteria and other kingdoms of life. This has been confirmed those last fifteen years as we have seen an explosion both in the number of regulatory RNAs identified and in our understanding of the variety of mechanims used by these molecules to control gene expression. Our studies range from the role of cis-elements of the target-mRNA in translational control by ribosomal proteins to the mechanisms of control exerted by cis- or trans-encoded antisense RNAs or the role of other actors, such as ribonucleases or the RNA chaperone Hfq. We are working with Escherichia coli, which is a model organism of choice for these studies given the knowledge that we have of this bacterium and the available tools to study it. But a lot of our findings are expected to be applicable to other pathogenic or non-pathogenic enterobacteria.
Theme n°2:
RNA maturation and stability
In E. col, RNA stability depends on endoribonucleolytic cleavages which initiate the process of degradation but also on the addition of poly(A) tails which facilitate the elimination of RNA fragments by exoribonucleases. Other actors, such as RNA helicases, ribosomes, Hfq protein and regulatory RNAs also participate in these processes. In spite of the fact that most (if not all) of the ribonucleases that mediate RNA maturation or decay have been identified, their individual functions are not well understood. One of our unexpected findings was that polyadenylation mediates bacterial RNA decay. Long regarded as a characteristic of eukaryotic mRNA, polyadenylation now appears to be a universal process for all classes of RNA. In both types of cells, this degradation pathway plays a role in quality control of RNA by degrading specific non-functional RNA. It also promotes the degradation of small RNA fragments generated by endoribonucleases during maturation or inactivation of the primary transcription allowing their degradation by exoribonucleases. However, the poly(A)-polymerase also modifies the functional stability of mRNA, thus demonstrating that polyadenylation can have a role in E. coli gene expression. The Hfq protein is involved in various mechanisms including RNA degradation and translation. This protein has an essential function after stress and participates in the expression of virulence factors in many pathogenic bacteria. It may act either directly on mRNA or indirectly by facilitating the interaction of mRNA with sRNAs. Interestingly, Hfq also modifies the abundance of certain mRNAs independently of their degradation. Moreover it has a role in the poly(A)dependent degradation pathway. We use in vivo and in vitro approaches to analyze the mode of action of Hfq and its function in cell metabolism. Our main objective is to understand how these various factors participate in RNA based regulation of gene expression.
Theme n°3:
small RNAs in cellular networks
Numerous studies point to a strong connection between sRNAs and other regulators. Indeed, not only is the synthesis of sRNAs highly regulated at the transcriptional level by transcription factors or alternative sigma factors, but many sRNAs also directly regulate synthesis of transcriptional regulators. The biology of the resulting mixed regulatory networks is still poorly understood. We are studying several examples that point out to a previously unsuspected complexity. For instance, two unrelated small RNAs that regulate synthesis of the PhoQ/PhoP two-component system by a similar mechanism have completely different effects on expression of the PhoP regulon. Together with the known competition between sRNAs for Hfq or between multiple targets of a given sRNA, our results highligt the requirement for systems biology when studying sRNA-mediated regulations.
