Regulated maturation and controlled degradation: the life cycle of RNAs in Haloferax volcanii

调控成熟和控制降解：Haloferax volcanii 中 RNA 的生命周期

• 批准号: 287106579
• 负责人: Professorin Dr. Anita Marchfelder
• 金额: --
• 依托单位: Institut für Molekularbiologie und Biotechnologie der Prokaryoten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/287106579?language=en
• 关键词: Regulated; maturation; controlled; degradation; life

Ribonucleases have a central role in the RNA metabolism of the cell. All RNAs are transcribed as precursor molecules that have to undergo a series of processing steps to generate the final mature molecule. In addition the cell has to remove in a controlled and regulated manner all RNAs, that have defects or are not required anymore. For these processes ribonucleases are essential and up to now only a few ribonucleases have been identified in Archaea. The goal of this project is to identify and characterize ribonucleases in the model archaeon Haloferax volcanii. In our preliminary work first data concerning three different ribonucleases have been acquired: data about the tRNase Z, the essential tRNA 3´ processing enzyme, and the RNase G/E as well as the protein RppH.In this proposal all of the biological functions of the following enzymes will be studied: tRNase Z, RNase G/E, RppH and tRNA splice endonuclease. Furthermore, using in vitro approaches new enzymes will be identified catalysing RNA 5´-> 3´-degradation, RNA 3´-> 5´-degradation and degradation of double stranded RNA. In addition, proteins that have been annotated as potential ribonucleases will be investigated. All experiments will be carried out in H. volcanii, since this archaeon is easy to genetically modify, the genome is sequenced and annotated, a reporter gene system is available and we are currently developing a knock down system for it in a parallel project. Recently a knock down system for prokaryotes has been developed (CRISPRi). This will allow to study and identify the biological function of essential genes. We will apply this method to study the essential ribonucleases tRNase Z and tRNA splice endonuclease.The analysis of ribonucleases in the frame of this project will provide information about RNA maturation and degradation in H. volcanii and thereby add new important data about the RNA metabolism in Archaea. New insights into the here studied RNases will also be interesting for the general RNA community since we will study homologs of bacterial and eukaryotic enzymes. Another interesting angle of this study is the evolutionary aspect. We expect to find next to the homologs to bacterial and eukaryotic enzyme also archaea-specific RNases, meaning that archaeal cells contain a mixture of RNases of all three domains. Comparison of these enzymes to each other and the analysis of interactions between them will be another interesting aspect of this study.

核糖珠在细胞的RNA代谢中具有核心作用。所有RNA均被转录为前体分子，必须进行一系列处理步骤以生成最终成熟分子。此外，该单元格必须以受控和调节的方式删除所有具有缺陷或不再需要的RNA。对于这些过程，核糖核酸是必不可少的，到目前为止，只有几个核糖核酸酶在古细菌中鉴定出来。在我们的初步工作中，该目标的目的是获得有关三个不同色带属的第一个数据：有关TRNase Z，必需的TRNA 3´加工酶和RNase g/e的数据数据以及蛋白质RPPH。此外，使用体外方法将确定新酶的催化RNA 5´-> 3´降解，RNA 3´-> 5´-降解和双链RNA的降解。此外，将研究已注释为潜在核糖珠的蛋白质。所有实验都将在火山谷链球菌中进行，因为该古迹易于基因修改，基因组进行了测序并注释，因此可以使用报告基因系统，并且我们目前正在并行项目中为其开发敲门系统。最近，开发了一种用于原核生物的敲门系统（CRISPRI）。这将允许研究和识别必需基因的生物学功能。我们将应用这种方法来研究必需的色带核酸酶TRNase Z和tRNA剪接核酸内切酶。该项目框架中的带核酸酶的分析将提供有关H. Volcanii中RNA成熟和降解的信息，从而添加有关Archaea中RNA代谢的新重要数据。对这里研究的RNass的新见解也将对一般的RNA社区感兴趣，因为我们将研究细菌和真核生物酶的同源物。这项研究的另一个有趣的角度是进化方面。我们希望在细菌和真核酶的同源物旁边找到古细菌特异性的RNase，这意味着古细胞包含所有三个结构域的RNase的混合物。这些酶相互比较以及它们之间的相互作用分析将是本研究的另一个有趣的方面。