Discovery and characterization of noncoding RNAs in prokaryotes

除了储存遗传信息外，DNA和RNA现在已知还有更多的功能。然而，更多的非编码RNA（ncRNA）经常被发现，一些工作表明我们可能只是触及了表面。我们将努力寻找更多的ncRNA，如核糖开关，这是ncRNA作为代谢受体，以控制基因。生物信息学、生物化学、微生物学和遗传学方法的组合将用于进一步表征所发现的RNA。该实验室追求两个主要的研究目标，以进一步了解ncRNA介导的细菌基因调控：1-发现ncRNA 1.1-通过寻找保守的基序与生物信息学; 1.2-与生物化学技术直接从细菌基因组中选择核糖开关。2-表征ncRNA 2.1-我们在Sinorhizobium melliloti（一种对植物有益的细菌物种）中发现的推定的钙敏感核糖开关; 2.2-我们发现的结合S-腺苷甲基化酶（SAM，在所有活生物体中发现的甲基化所必需的辅因子）的核糖开关的变体; 2.3-我们发现的推定的胍核糖开关; 2.4-我们将在目标1中发现的基序。 该实验室为发现新的ncRNA建立了工具。例如，Ribogap数据库与寻找新RNA结构的管道相结合，使我们能够发现功能相关基因（例如Ca 2+泵和Ca 2+调节因子）前面保守的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（例如Ca 2+传感核糖开关），并且已经被证明是成功的。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ncRNA，包括没有相同偏见的生物信息学方法。与此同时，我们将使用生物化学筛选技术来寻找参与基因调控的其他ncRNA。我们开发的技术，SR-PAGE，将使我们能够选择核糖开关对多个配体同时进行。 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靶分子的相互作用; ii）体内技术，以研究核糖开关如何调节基因; iii）用i）和ii）分析核糖开关的各种突变形式，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钙核糖开关，以及我们期望通过我们的生物信息学管道和SR-PAGE找到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基因控制的机制，以及进化如何修补RNA结构，以优化它们的特定功能和环境。

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.