Small regulatory RNAs from the halophilic archaeon Haloferax volcanii

在前期工作中，我们利用RNomics和High Throughput Sequencing等实验方法和生物信息学分析对火山盐铁古菌进行了候选sRNAs的鉴定。仅在实验方法的基础上，已经鉴定出145个候选基因间sRNAs和45个反义sRNAs。为了研究基因间sRNAs的生物学功能，已经制作了32个缺失菌株。通过过表达反义sRNA基因来分析反义sRNA的功能，因为在这种情况下，菌株不能产生缺失。此外，已经开展了鉴定sRNA分子靶标的第一个方法。下一个资助期的目标是(1)确定基因间sRNA的靶标，(2)详细描述sRNA-靶标相互作用，以确定古细菌sRNA调控机制，(3)分析tRNA衍生片段（trf）的潜在调控作用，(4)研究其他sRNA，以进一步确定靶标，(5)分析Lsm蛋白对sRNA功能的影响。本项目的主要重点是确定sRNA靶点并详细研究它们与sRNA的相互作用。几种方法将用于鉴定sRNA靶标。我们将使用sRNA基因缺失菌株与野生型菌株的转录组比较。此外，还将对sRNA基因进行脉冲过表达，并在过表达前后进行转录组比较。这两种方法都将揭示哪些基因在缺失菌株和过表达菌株中分别被上调和下调。此外，我们将对选定的调控rna进行标记实验，以确定它们的靶标（蛋白质靶标和rna靶标）。确定目标的第三种方法是生物信息学方法。靶RNA和sRNA之间的相互作用可以立即分析顺式编码的反义RNA，这些RNA位于其靶标的正对面。asrna -靶RNA对将使用凝胶移位、突变方法和报告基因系统进行详细分析。基因间sRNAs与其靶标相互作用的分析将使用与asrna相同的方法进行。对于这两种sRNA类型，将分析目标mrna 3´- utr上的主要相互作用，因为这种类型的相互作用不同于迄今为止在细菌中观察到的sRNA相互作用模式。

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.