Characterization of three novel RNAi machinery components in C. elegans

RNA interference (RNAi) is a homology-based and antisense small RNA-dependent gene silencing mechanism found in most eukaryotes. This dissertation describes identification and characterization of three novel RNAi pathway components in nematode Caenorhabditis elegans: rde-10, rde-11 and rde-12. We demonstrated that they are required for targeted mRNA degradation and the subsequent secondary siRNA biogenesis.;The molecular mechanisms for targeted mRNA degradation in C. elegans undergoing RNA interference (RNAi) are not fully understood. Using a combination of genetic, proteomic and biochemical approaches, we have identified a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary short interfering RNA (siRNA). Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Results of deep sequencing analyses reveal a 5-fold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, despite normal primary siRNA and micro-RNA biogenesis. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. .;RDE-12, a putative DEAD-box RNA helicase, was identified from the same genetic screen. A GFP::RDE-12 fusion protein forms cytoplasmic foci in somatic cells and resides in peri-nuclear P granules in the germline. RDE-12 engages target mRNAs in response to RNAi and is required for the secondary siRNA biogenesis. We hypothesize that RDE-12 acts as a linkage between mRNA targeting and secondary siRNA synthesis in the RNAi pathway.