Discovery and characterization of noncoding RNAs in prokaryotes

除了存储遗传信息外，DNA和RNA现在还具有更多的功能。然而，经常发现更多的非编码RNA(NcRNAs)，一些研究表明，我们可能只触及了皮毛。我们将努力寻找更多的ncRNAs，如核糖开关，这是作为代谢物受体的ncRNAs，从而控制基因。生物信息学、生化、微生物学和遗传学方法的结合将被用来进一步确定发现的RNA的特征。该实验室追求两个主要研究目标，以加深我们对ncRNA介导的细菌基因调控的理解：1-发现ncRNAs 1.1-通过寻找保守基序的生物信息学和1.2-利用生化技术直接从细菌基因组中选择核糖开关。2-鉴定ncRNAs2.1-我们在中华根瘤菌(一种对植物有益的细菌物种)中发现的可能的钙敏感核糖开关；2.2-我们发现的与S结合的核糖开关的变体-腺苷蛋氨酸(SAM，一种在所有生物中都存在的甲基化必需的辅因子)；2.3-我们发现的假定的鸟苷核糖开关；2.4-我们将在目标1中找到的基序。*实验室建立了发现新型ncRNAs的工具。例如，RiboGap数据库与寻找新的RNA结构的管道相结合，使我们能够发现在功能相关基因(如钙泵和钙调节因子)之前保守的RNA结构。这些有针对性的计算搜索旨在发现可能具有与给定功能相关的调节作用的ncRNA(如钙感应核糖开关)，并已被证明是成功的。我们将继续使用我们的结构发现生物信息学管道来寻找与SAM、胍和信使分子相关的ncRNAs，包括使用没有相同偏见的生物信息学方法。同时，我们将利用生化筛选技术寻找其他参与基因调控的ncRNAs。我们开发的技术SR-PAGE将允许我们同时选择针对多个配体的核糖开关。我们研究核糖开关的主要实验工具包括：i)体外技术，以精确研究核糖开关与其目标分子的相互作用；ii)体内技术，研究核糖开关如何调节基因；iii)各种突变版本的核糖开关，同时分析i)和ii)，以帮助我们破译导致基因表达变化的“结构转换”机制。我们将使用这些方法来研究SAM-核糖开关的许多变体，我们发现的新的推定的钙核糖开关，以及我们希望在我们的生物信息学管道和SR-PAGE中发现的所有其他核糖开关。通过更详细地研究这些，我们将更好地了解细菌中的基因控制机制，以及进化是如何改变RNA结构以优化它们以适应特定功能和环境的。

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.