The control of mRNA half-life is an important, but often under-estimated, way of regulating gene expression. Although the processes of mRNA maturation and degradation are well-understood in Escherichia coli, this is not yet the case in B. subtilis. This research project seeks to bring our level of understanding of these processes in B. subtilis up to par with that of E. coli
This project focuses on four main research areas (I) the identification of new ribonucleases in B. subtilis (2) the characterisation of the roles of two orphan RNases, i.e. whose substrates are not known (3) the characterisation of the roles of previously identified RNases in mRNA degradation pathways and (4) the resolution of the crystal structure of the most important RNases, if possible in complex with their substrates
The techniques employed range from bioinformatics to crystallography, and include genetic, biochemistry and molecular biology. The project also exploits high resolution transcriptome analysis to identify transcripts affected in mutants for the most important RNases
The major results of the project to date are (1) the resolution of the crystal strucutre of RNase J complexed to RNA (2) the identification of the RNA pyrophosphohydrolase RppH, important for the deprotection of RNAs, by removing the 5' triphosphate of primary transcripts to a 5' monophosphate (3) the identification of the substrates of three essential RNases, RNase J1, Y and III, on a global level by high resolution transcriptome analyses
The high resolution transcriptome analysis allowed us to identify several small regulatory RNAs and the enzymes involved in their turnover. These results pave the way for an important aspect of the project, the control of RNA degradation by regulatory RNAs coded both in cis and in trans relative to their targets
The publication of three original papers in Structure, Molecular Cell and PLoS Genetics is testament to the state progress of the project. We have also published two review articles, one in Curr. Opin. Microbiol. and the other as a chapter in an edition o
Our vision of RNA metabolism in bacteria and in eukaryotes comes largely from studies in Escherichia coli and Saccharomyces cerevisiae, respectively. While these two organisms remain the models of reference in this domain, it has been clear for some time that S. cerevisiae is not representative of all eukaryotes and, much more recently, that E. coli only provides a restricted view of the bacterial world, primarily as a result of studies in the Gram-positive Bacillus subtilis.
E. coli and B. subtilis have very different pathways for many fundamental cellular processes, the result of over a billion years of independent evolution. The pathways of RNA maturation and degradation are excellent examples of this differentiation. Only eight of the >30 ribonucleases (RNases) identified in these two organisms are common to both. Even the essential RNases are different. The chemistry of the turnover reactions is not the same, nor are the mechanisms of initiating degradation. In our previous ANR project, we discovered that B. subtilis possesses a 5’-3’ exoribonuclease activity (provided by RNase J1), a property that was previously thought to be confined to eukaryotes. We also learned from these studies that some important enzymes have yet to be identified in B. subtilis and that other suspected RNases lack an associated function. Most of these enzymes are unique to B. subtilis. Despite the few missing enzymes, we believe that we have most of the tools necessary to perform an in-depth analysis of B. subtilis RNA degradation pathways. By answering new questions raised during our previous ANR project, we hope to bring our understanding of RNA metabolism in B. subtilis much closer to the levels currently enjoyed by E. coli and S. cerevisiae.
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B (Divers public)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
Help of the ANR 400,000 euros
Beginning and duration of the scientific project: - 48 Months