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

真核RNA依赖性RNA聚合酶（RdRPs）利用RNA模板合成互补RNA分子，参与多种基因调控过程。我们已经证明，三个RdRP从阿米巴D。discoideum参与调节两个反转录转座子。在这个项目中，我们将分析这些酶的生化和功能细节。过度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。在只有一个剩余RdRP的突变菌株中，我们将在深度测序方法中定义该酶的内源性siRNA产物。蛋白质相互作用伴侣将从亲和纯化中鉴定，并将有助于与深度测序数据一起定义RdRP参与的细胞途径。去除任一聚合酶对由反转录转座子序列组成的网囊藻着丝粒的组织和维持的影响将通过各种方法在各自的敲除菌株中进行分析。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分析RdRPs在转录、转录后或翻译水平上对基因沉默的贡献。

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.