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

在rna介导的干扰（RNAi）中，产生小的非编码rna （sirna）来指导特定的基因沉默，因为它们编程各种效应复合物来降解同源信使rna，或沉默其转录。许多rnai样途径在植物、真菌、酵母和动物体内发挥内源性作用。内源性（内端）RNAi是指sirna从内源性位点衍生并收敛回内源性位点的机制。虽然endoRNAi的基因沉默步骤越来越清楚，但对它们的起始和特异性知之甚少。RNA依赖的RNA聚合酶（RdRP）往往是起始的endonai的核心。有了这笔资助，我们将研究RRF-3 RdRP，以破译其特异性的基础，并阐明ERI endoRNAi启动的机制。

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.