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和高通量测序）和生物信息学分析在模型Archaeon Haloferax火山中鉴定候选SRNA。基于实验方法，已经确定了145个候选基因间SRNA和45个反义SRNA。为了研究基因间SRNA 32缺失菌株的生物学功能。通过过表达反义SRNA基因，可以分析反义SRNA的功能，因为在这种情况下无法删除菌株。此外，已经采用了第一个鉴定SRNA分子靶标的方法。下一个资金期限的目标是（1）识别基因间SRNA的目标，（2）SRNA-TARGET互动的详细表征能够确定细分SRNA调控的机制，（3）对TRNA衍生的片段的潜在调节作用的分析（TRFS）的潜在调节作用（trfs），（4）允许对（4）识别的识别（4）相互识别（4）相同的相互作用（4）及其及其识别及其（4）关于SRNA功能的蛋白质。该项目的主要重点是确定SRNA目标并详细研究其与SRNA的相互作用。将使用几种方法来识别SRNA靶标。我们将使用具有野生型菌株的SRNA基因缺失菌株的转录组比较。另外，将进行SRNA基因的脉冲过表达以及在过表达之前和之后的后续转录组比较。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目标）。识别目标的第三种方法将是一种生物信息学方法。可以立即分析靶RNA与SRNA之间的相互作用，分别针对位于其目标对面的顺式编码的反义RNA。将使用凝胶移位，突变方法和报告基因系统详细分析Asrna-Target RNA对。使用与ASRNA相同的方法进行基因间SRNA与目标的相互作用分析。对于两种sRNA类型，将分析目标mRNA 3'-UTRS的最首要相互作用，因为这种类型的相互作用与迄今在细菌中观察到的SRNA相互作用模式不同。