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

核糖核酸酶在细胞的RNA代谢中具有核心作用。所有的RNA都被转录为前体分子，这些前体分子必须经历一系列的加工步骤以产生最终的成熟分子。此外，细胞必须以受控和调节的方式去除所有有缺陷或不再需要的RNA。对于这些过程，核糖核酸酶是必不可少的，到目前为止，只有少数核糖核酸酶已被确定在Escherichia。该项目的目标是识别和表征模式古菌Haloferax volcano ii中的核糖核酸酶。在我们的初步工作中，已经获得了关于三种不同核糖核酸酶的第一批数据：关于tRNase Z（必需的tRNA 3 ′加工酶）和RNase G/E以及蛋白质RppH的数据。在本提案中，将研究以下酶的所有生物学功能：tRNase Z、RNase G/E、RppH和tRNA剪接核酸内切酶。此外，使用体外方法，将鉴定催化RNA 5 ′-> 3 ′-降解、RNA 3 ′-> 5 ′-降解和双链RNA降解的新酶。此外，将研究已注释为潜在核糖核酸酶的蛋白质。所有实验均在H. volcanii，因为这种古菌很容易遗传修饰，基因组被测序和注释，报告基因系统是可用的，我们目前正在开发一个敲除系统，它在一个平行的项目。最近已经开发了用于原核生物的敲除系统（CRISPRi）。这将允许研究和鉴定必需基因的生物学功能。我们将利用这种方法对核糖核酸酶tRNase Z和tRNA剪接核酸内切酶进行研究，为进一步了解H. volcanii，从而增加了新的重要数据的RNA代谢在美洲。新的见解在这里研究的RNA酶也将是有趣的一般RNA社区，因为我们将研究同源的细菌和真核生物酶。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生物酶的同源物旁边还发现古细菌特异性RNA酶，这意味着古细菌细胞含有所有三个结构域的RNA酶的混合物。这些酶相互之间的比较和它们之间的相互作用的分析将是本研究的另一个有趣的方面。

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.