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

理解反义：内源性 RNAi 中 RNA 依赖性 RNA 聚合酶的活性

• 批准号: 341457-2012
• 负责人: Duchaine, Thomas
• 金额: $ 2.48万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=572262
• 关键词: Making; sense; antisense; activities; RNA

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.

在RNA介导的干扰（RNAi）中，产生小的非编码RNA（siRNA）以指导特定的基因沉默，因为它们编程多种效应复合物以降解同源信使RNA或沉默其转录。许多RNAi样途径在植物、真菌、酵母和动物中内源性发挥作用。内源性（endo）RNAi是指其中siRNA来源于内源基因座并会聚回内源基因座的机制。虽然endoRNAi的基因沉默步骤变得越来越清楚，但对它们的起始以及基于它们的特异性知之甚少。RNA依赖性RNA聚合酶（RdRP）通常是endoRNAi起始的核心。有了这笔资金，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。 具体目标：1-为了在体内表征RdRP的底物和产物，我们将使用交联、免疫沉淀和深度测序（HITS-CLIP和PAR-CLIP）鉴定RRF-3相关RNA。我们将利用RdRP模块组件的一组重要的无义和错义等位基因来评估测定的特异性，区分底物与产物，并解读它们在endoRNAi中的含义。2-为了鉴定RdRP特异性的决定因素，我们将检查RRF-3 RdRP及其产物在无细胞胚胎系统中的RNA底物内的要求。源自影响ERI endoRNAi机制的突变体的提取物将用于控制特异性，并使ERI endoRNAi起始的多个步骤去卷积。 3-最后，将使用概括的杆状病毒表达复合物来表征RRF-3的活性和特异性。 RdRPs是RNAi最古老的组分之一。该项目将阐明RNAi的机制和功能多样性的分子基础。