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

• Applicant: Professor Dr. Christian Hammann
• Subject Area: Cell Biology
• Term: from 2011 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 192916274

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.

• DFG Programme: Research Grants