Small regulatory RNAs from the halophilic archaeon Haloferax volcanii

来自嗜盐古菌 Haloferax volcanii 的小调控 RNA

• 批准号: 238237704
• 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: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/238237704?language=en
• 关键词: Small; regulatory; RNAs; halophilic; archaeon

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.

在初步工作中，使用实验（RNomics和高重复序列测序）和生物信息学分析在模式古菌Haloferax volcanii中进行候选sRNA的鉴定。仅在实验方法的基础上，已经鉴定了145个候选基因间sRNA和45个反义sRNA。为了研究基因间sRNA的生物学功能，已经制备了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，因为在这种情况下不能产生缺失菌株。此外，已经采取了鉴定sRNA分子的靶标的第一种方法。下一个资助期的目标是（1）鉴定基因间sRNA的靶点，（2）详细描述sRNA-靶点相互作用，以便能够确定古细菌sRNA调控的机制，（3）分析tRNA衍生片段（tRFs）的潜在调控作用，（4）研究其他sRNA，以进一步鉴定靶点，和（5）分析Lsm蛋白的sRNA功能。该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。将使用几种方法来鉴定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，将进行sRNA基因的脉冲过表达和随后的过表达前后的转录组比较。这两种方法将分别揭示哪些基因在缺失菌株和过表达菌株中上调和下调。此外，我们将使用选定的调控RNA进行标记实验，以识别它们的靶标（蛋白质和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，其直接位于其靶的对面。将使用凝胶位移、突变方法和报告基因系统详细分析asRNA-靶RNA对。将使用与asRNA相同的方法分析基因间sRNA与其靶标的相互作用。对于这两种sRNA类型，将分析靶mRNA 3 ′-UTR处的最重要的相互作用，因为这种类型的相互作用不同于迄今在细菌中观察到的sRNA相互作用模式。