Bacillus subtilis as an alternative model of RNA maturation and decay – subtilRNA2
Studies on the pathways of RNA maturation and degradation in the Gram positive model organism Bacillus subtilis
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
Identification and characterisation of new ribonucleases in B. subtilis : the enzymes, their substrates and their structures
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 identification of the RNA pyrophosphohydrolase RppH, that deprotects RNAs by converting the 5' triphosphate of primary transcripts to a 5' monophosphate (2) the resolution of the crystal structures of RNase J, RNase Z and RppH complexed to RNA (3) the identification of the substrates of three key RNases, RNase J1, Y and III, on a global level by tiling array analyses and (4) the study of the RNase III-dependent regulation of the txpA toxin mRNA by the antisense RNA ratA in B. subtilis.
We are currently working on 3 major remaining questions (1) the roles of the two orphan ribonucleases KapD and YacP (2) the identification of the enzyme that matures the 3' end of 16 rRNA and (3) the role of transacting regulatory RNAs in mRNA turnover in B. subtilis.
Seven original papers in Molecular Cell, J. Bacteriol, PNAS, and 2 each in PLoS Genetics and Structure. We have also published three review articles, in RNA Biol, Curr. Opin. Microbiol. and as a chapter in an edition of Nucleic Acids and Molecular Biology dedicated to ribonucleases
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.
Monsieur Ciaran CONDON (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – firstname.lastname@example.org
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.
CNRS 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