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

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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.