Activity and function of RNA-dependent RNA polymerases of Dictyostelium discoideum

盘基网柄菌 RNA 依赖性 RNA 聚合酶的活性和功能

• 批准号: 192916274
• 负责人: Professor Dr. Christian Hammann
• 金额: --
• 依托单位: HMU Health and Medical University Potsdam
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/192916274?language=en
• 关键词: Activity; function; RNA; dependent; polymerases

Eukaryotic RNA-dependent RNA polymerases (RdRPs) use RNA templates in the synthesis of com-plementary RNA molecules and participate thereby in various gene regulatory processes. We have shown that the three RdRPs from the amoeba D. discoideum are involved in the regulation of two retrotransposons. In this project we will analyse these enzymes in biochemical and functional detail. Over-expressed proteins will be used to define substrates and products, and to clarify mechanistic questions of their reactions, including whether RNA primers are required. In mutant strains with only one remaining RdRP, we will define this enzyme’s endogenous siRNA products in deep sequencing approaches. Protein interaction partners will be identified from affinity purification and will help to define, together with the deep sequencing data, cellular pathways, in which the RdRPs are involved. The effect that the removal of either polymerase has on the organisation and maintenance of the Dictyostelium centromers, which are made up by retrotransposon sequences, will be analysed by various approaches in the respective knock out strains. Finally, RNA expression vectors will be tested for their silencing capacity. Using different techniques, we thus will analyse the contribution of the RdRPs to gene silencing on the transcriptional, post-transcriptional or translational level.

真核RNA依赖性RNA聚合酶（RdRP）在互补RNA分子的合成中使用RNA模板，从而参与各种基因调控过程。我们已经证明来自盘状变形虫的三个 RdRP 参与两个逆转录转座子的调节。在这个项目中，我们将详细分析这些酶的生化和功能。过表达的蛋白质将用于定义底物和产物，并澄清其反应的机制问题，包括是否需要 RNA 引物。在仅剩余一个 RdRP 的突变菌株中，我们将通过深度测序方法定义该酶的内源 siRNA 产物。蛋白质相互作用伙伴将通过亲和纯化来鉴定，并将与深度测序数据一起帮助定义 RdRP 所涉及的细胞途径。将通过各种方法在各自的敲除菌株中分析去除任一聚合酶对盘基网柄菌着丝粒（由反转录转座子序列组成）的组织和维持的影响。最后，将测试 RNA 表达载体的沉默能力。因此，我们将使用不同的技术在转录、转录后或翻译水平上分析 RdRP 对基因沉默的贡献。