Making sense of antisense: the activities of RNA-dependent RNA polymerases in endogenous RNAi

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In RNA-mediated interference (RNAi), small non-coding RNAs (siRNAs) are generated to direct specific gene silencing as they program a variety of effector complexes to degrade cognate messenger RNAs, or silence their transcription. Many RNAi-like pathways function endogenously in plants, fungi, yeast, and animals. Endogenous (endo)RNAi refers to mechanisms wherein siRNAs are derived from and converge back on endogenous loci. While the gene silencing steps of endoRNAi are becoming clear, little is known about their initiation, and on the basis of their specificity. RNA-dependent RNA polymerases (RdRP) are often central to initiation of endoRNAi. With this grant, we will examine the RRF-3 RdRP to decipher the basis for its specificity, and elucidate the mechanism of ERI endoRNAi initiation. Specific aims: 1- To characterize the substrate and product of the RdRPs in vivo, we will identify RRF-3-associated RNAs using cross-linking, immunoprecipitation, and deep sequencing (HITS-CLIP and PAR-CLIP). We will take advantage of an important battery of nonsense and mis-sense alleles of the RdRP module components to assess the specificity of the assay, to discriminate the substrates from the products, and to decipher their implication in endoRNAi. 2- To identify the determinant(s) of the RdRP specificity, we will examine the requirements within the RNA substrates of the RRF-3 RdRP, and its products in an cell-free embryonic system. Extracts derived from mutants affecting the ERI endoRNAi machinery will be used to control for specificity, and de-convolute the mutliple steps of ERI endoRNAi initiation. 3- Finally, the activity and specificity of RRF-3 will be characterized using recapitulated baculovirus-expressed complexes. RdRPs are amongst the most ancient components for RNAi. This project will clarify the molecular basis for the mechanistic, and functional diversification of this ill-understood key component of RNAi.