Friday, 4th October, 2024
Tomas Caetano will present their research on the study of the regulation of the dual degradation activity of RNase J by epi-transcriptomic modifications and secondary structures.
Abstract :
Ribonucleases responsible of RNA degradation and/or maturation are vital for the cell’s adaptation to sudden environmental changes. They have two modes of action, an endonucleolytic mode cleaving RNAs internally, and an exonucleolytic mode degrading them from their 5′ or 3′ ends. Endoribonucleases reflect a remarkable case of convergent evolution, as this activity is carried by RNase E, RNase Y and RNase J, proteins with no sequence similarity. It has also been shown that, depending on the bacterial species, the substrates and activity of these ribonucleases can differ, making them sometimes essential for cell survival. My thesis work focused on the role and regulation of the RNase J complex in the pathogenic bacteria Staphylococcus aureus. This complex has dual endo- and 5′ exoribonucleolytic activity. The latter shows a preference for 5′ monophosphorylated RNAs (5’P) over triphosphorylated RNAs (5’PPP). During my thesis work, I developed statistical and bioinformatics methods adapted to the processing and analysis of RNA 5′-end sequencing data obtained by a collaborating team who had developed experimental protocols to study the activity of the RNase J complex in vivo and on a genome-wide scale. These developments have been integrated into an open- source R package accessible on the CNRS GitLab instance. The first question addressed was the identification of the NUDIX ribopyrophosphohydrolase (RppH) protein(s) responsible for converting 5’PPP RNAs to 5’P for degradation by the RNase J complex. Of the five RppHs predicted in silico in S. aureus, only the one most similar to that of B. subtilis has been published, concluding that it has little impact on RNA stability. My work has validated the experimental protocol in terms of its ability to translate abundances into proportions of the phosphorylation states of each RNA in order to statistically identify differences in proportions. Deletion mutants for the five RppH candidates (single or combined deletions) were obtained. None of the mutants studied totally lost RppH activity. The regulatory system for this mechanism is therefore more complex than expected in S. aureus, and the difficulty to obtain certain deletion mutants suggests a particular mechanism in this organism.
In parallel, I exploited previously available RNA-Seq data obtained by blocking transcription and measuring RNA abundance over time by transcriptomic analyses, between a wild-type S. aureus strain and a ΔRNase J strain. By comparing RNA stability, I showed that 47% of genes had RNAs whose degradation depended on this enzyme, with a preference for highly expressed genes.
I also carried out a quantitative analysis of degradation intermediates relative to open reading frames, obtained using another method for in vivo quantification of RNA 5′ ends. In the wild-type strain, I highlighted a periodic distribution of 3 nucleotides corresponding to codons, which is practically no longer observed in the ΔRNase J mutant. I thus demonstrated that RNase J can follow the last ribosome during translation, and that it is a major player in the co-translational degradation mechanism. Further analysis of the same data enabled me to predict 125 endoribonucleolytic cleavage sites for RNase J. These preliminary results may serve as a starting point for further studies. My work as a whole has therefore provided new information on the in vivo activity and role of the RNase J complex in S. aureus, and the methods I have developed dedicated to the analysis of 5′ RNA-Seq data may be used in other studies of this type.
The thesis defense will be preceded by a seminar : Dr Anaïs Le Rhun (INSERM – Université de Bordeaux) : « The bacterial hok/Sok toxin-antitoxin system : regulation & function » .