Discovery and characterization of noncoding RNAs in prokaryotes

除了存储遗传信息外，现在已知 DNA 和 RNA 还具有更多功能。然而，更多的非编码 RNA (ncRNA) 经常被发现，一些研究表明我们可能只触及了表面。我们将努力寻找更多的 ncRNA，例如核糖开关，它们是充当代谢受体来相应控制基因的 ncRNA。将结合生物信息学、生物化学、微生物学和遗传学方法来进一步表征所发现的 RNA。该实验室追求两个主要研究目标，以进一步了解细菌中 ncRNA 介导的基因调控： 1-通过生物信息学寻找保守基序，发现 ncRNA 1.1； 1.2-利用生化技术直接从细菌基因组中选择核糖开关。 2-表征ncRNA 2.1-我们在中华根瘤菌（一种对植物有益的细菌）中发现的假定的钙感应核糖开关； 2.2- 我们发现的结合 S-腺苷甲硫氨酸的核糖开关变体（SAM，一种在所有生物体中发现的甲基化必需的辅助因子）； 2.3-我们发现的假定的胍核糖开关； 2.4-我们将在目标 1 中找到的基序。实验室构建了用于发现新型 ncRNA 的工具。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 Ca2 泵和 Ca2 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（例如 Ca2 感应核糖开关）相关的调节作用的 ncRNA，并且已被证明是成功的。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 ncRNA，包括使用不具有相同偏差的生物信息学方法。与此同时，我们将使用生化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) 精确研究核糖开关与其目标分子相互作用的体外技术； ii) 体内技术，研究核糖开关如何调节基因； iii) 使用 i) 和 ii) 分析核糖开关的各种突变版本，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-核糖开关的众多变体、我们发现的新的假定钙核糖开关，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的基因控制机制，以及进化如何修补 RNA 结构以优化它们以适应特定的功能和环境。

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.