Marek Malicki

• About 10% of the compact genome of Dictyostelium discoideum comprise of transposable elements (TEs). The active TEs, are highly mutagenic, and they potentially negatively impact the fitness of their host. Dictyostelium’s RNAi machinery and their components, serve as a toolkit indispensable to tightly regulate the activity of TEs. In a recent study, it has been shown that out of three identified RdRPs in the social amoeba (RrpA, RrpB and RrpC), only RrpC plays an important role in the silencing of the most prominent retrotransposon, DIRS-1. Laser scanning microscopy analysis of cells overexpressing RdRP GFP-fusion proteins allowed to describe RrpA and RrpB as nuclear proteins co-localizing with the triple methylated lysine on histone 3 and HcpA, a homologue of Heterochromatin protein 1. In contrast to RrpA and RrpB, the RrpC was found exclusively in the cytoplasm, which correlates with previous findings that DIRS-1-related siRNAs accumulate in the same cellular compartment. The extensive proteomic study of RdRPs, on the side, led to the development of a method to purify the endogenous AgnA protein, a putative interaction partner of RrpC. Moreover, it has been shown, that AgnA binds 21 nt long RNAs. In all likelihood these small RNAs direct the putative complex, formed by RrpC and AgnA, later called Retrotransposon Silencing Complex (RSC) to their targets. Moreover, both parts of the putative RSC plays a crucial role in silencing of DIRS-1 and Skipper. The retrotransposition competency of DIRS-1 and Skipper was monitored by genetically traceable synthetic TEs used in retrotransposition assays. Extending the observation that DIRS-1 and Skipper are silenced by post-transcriptional gene silencing trough the RSC activity, this thesis provides also first indications that Skipper, is also repressed at the level of chromatin via transcriptional gene silencing. This process, RNA-mediated heterochromatisation, appears to involve the activity of RrpA.