Small regulatory RNAs from the halophilic archaeon Haloferax volcanii

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In the preliminary work the identification of candidate sRNAs was carried out in the model archaeon Haloferax volcanii using experimental (RNomics and High Throughput Sequencing) and bioinformatics analyses. On the basis of experimental approaches alone 145 candidate intergenic sRNAs and 45 antisense sRNAs have been identified. For the investigation of the biological function of the intergenic sRNAs 32 deletion strains have already been made. The function of the antisense sRNAs was analysed by overexpressing the antisense sRNA genes since in this case deletions strains can not be made. In addition, first approaches to identify targets for sRNA molecules have been undertaken. The goals for the next funding period are (1) the identification of targets for the intergenic sRNAs, (2) the detailed characterisation of sRNA-target interaction to be able to determine the mechanism of archaeal sRNA regulation, (3) analysis of the potential regulatory role of tRNA derived fragments (tRFs), (4) investigation of additional sRNAs which will allow further identification of targets, and (5) analysis of the Lsm protein in respect to sRNA function. The main focus of this project is to identify sRNA targets and to study their interaction with sRNAs in detail. Several methods will be used to identify sRNA targets. We will use transcriptome comparisons of sRNA gene deletion strains with wild type strains. In addition pulse overexpression of sRNA genes and subsequent transcriptome comparison before and after overexpression will be carried out. Both approaches will reveal which genes are up- and downregulated in the deletion strains and overexpression strains, respectively. Further, we will perform tagging experiments with selected regulatory RNAs to identify their targets (protein- as well as RNA-targets). A third approach to identify targets will be a bioinformatics approach. The interaction between target RNA and sRNA can immediately be analysed for the cis-encoded antisense RNAs, which are located directly opposite their target. The asRNA-target RNA pairs will be analysed in detail using gel shifts, mutational approaches and reporter gene systems. Analysis of interaction of intergenic sRNAs with their targets will be done using the same methods as for the asRNAs. For both sRNA types, foremost interactions at target-mRNA 3´-UTRs will be analysed since this type of interactions is different from the sRNA interaction modes hitherto observed in bacteria.