Discovery and characterization of noncoding RNAs in prokaryotes

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Aside from storing hereditary information, DNA and RNA are now known to have many more functions. Yet, more noncoding RNAs (ncRNAs) are regularly found and some work suggests that we may have only scratched the surface. We will work towards finding more ncRNAs such as riboswitches, which are ncRNAs that act as metabolite receptors to control genes accordingly. A combination of bioinformatics, biochemical, microbiological and genetic approaches will be used to further characterize the discovered RNAs. The laboratory pursues two main research goals to further our understanding of ncRNA-mediated gene regulation in bacteria:******1-Discover ncRNAs 1.1-with bioinformatics by looking for conserved motifs and; 1.2-with a biochemical technique to select riboswitches directly from bacterial genomes.******2-Characterize ncRNAs 2.1-the putative calcium-sensing riboswitch that we found in Sinorhizobium melliloti (a bacteria species beneficial to plants); 2.2- the variants we found of riboswitches that bind S-AdenosylMethionine (SAM, a cofactor found in all living organisms essential for methylation); 2.3-the putative guanidine riboswitches that we found and; 2.4-the motifs that we will find in objective 1.****** The laboratory built tools for the discovery of novel ncRNAs. For instance, the Ribogap database combined with a pipeline to find novel RNA structure, allows us to uncover RNA structures conserved in front of functionally-related genes (e.g. Ca2+ pumps and Ca2+-regulatory factors). These targeted computational searches aim at uncovering ncRNAs likely to have regulatory roles connected to the given functions (such as a Ca2+-sensing riboswitch) and have already been proven successful. We will continue to use our structure discovery bioinformatics pipeline to look for ncRNAs associated with SAM, guanidine and messenger molecules, including with bioinformatics approaches that do not have the same biases. In parallel, we will use a biochemical screening techniques to find other ncRNAs involved in gene regulation. The technique we developed, SR-PAGE, will allow us to select riboswitches against multiple ligands simultaneously.****** Our main experimental tools to study riboswitches include i) in-vitro techniques to precisely study the interaction of riboswitches with their target molecule; ii) in-vivo techniques, to study how riboswitches regulate genes and; iii) various mutant versions of the riboswitches analysed with both i) and ii) to help us decipher the mechanisms of “structure-switching” leading to changes in gene expression. We will use these methods to study the numerous variants of SAM-riboswitches, the new putative calcium riboswitch we discovered, as well as all the additional riboswitches we expect to find with our bioinformatics pipeline and SR-PAGE. By studying these in more details, we will better understand mechanisms of gene control in bacteria and how evolution tinkers with RNA structures to optimize them for particular functions and contexts.