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.

在前期工作中，通过实验（RNomics 和高通量测序）和生物信息学分析，在模型古菌 Haloferax volcanii 中鉴定了候选 sRNA。仅基于实验方法，已鉴定出 145 种候选基因间 sRNA 和 45 种反义 sRNA。为了研究基因间 sRNA 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，因为在这种情况下不能产生缺失菌株。此外，已经采取了第一种方法来识别 sRNA 分子的靶标。下一个资助期的目标是 (1) 识别基因间 sRNA 的靶标，(2) sRNA-靶标相互作用的详细表征，以便能够确定古菌 sRNA 调节的机制，(3) 分析 tRNA 衍生片段 (tRF) 的潜在调节作用，(4) 研究其他 sRNA，以进一步识别靶标，以及 (5) 分析 Lsm 与 sRNA 功能有关的蛋白质。该项目的主要重点是识别 sRNA 靶标并详细研究它们与 sRNA 的相互作用。将使用多种方法来识别 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，还将进行sRNA基因的脉冲过表达以及随后的过表达之前和之后的转录组比较。两种方法都将分别揭示哪些基因在缺失菌株和过度表达菌株中上调和下调。此外，我们将使用选定的调节 RNA 进行标记实验，以识别其靶标（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。可以立即分析靶标 RNA 和 sRNA 之间的相互作用，以找出顺式编码的反义 RNA，这些反义 RNA 位于其靶标正对面。 asRNA-靶标RNA对将使用凝胶位移、突变方法和报告基因系统进行详细分析。基因间 sRNA 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，将分析靶 mRNA 3'-UTR 处最重要的相互作用，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。