Investigating the Ago Cleavage Independent Mechanism(s) of RNAi

描述（由申请人提供）：短干扰RNA （sirna）是一种小RNA调节剂，能够在称为RNA干扰（RNAi）的过程中降低其靶mrna的蛋白质表达。RNAi对正常的细胞生理和适应非常重要，也是科学家们宝贵的遗传工具和临床治疗的一个有前途的来源。虽然典型的RNAi模型以sirna为导向的Argonaute蛋白（Agos）切割靶mRNA为特征，但最近的研究表明，ago介导的切割对于靶mRNA的抑制是必不可少的。其他研究和与微rna的比较表明，sirna可能通过ago依赖的翻译抑制和/或通过非ago核酸酶刺激降解来抑制其靶mrna。为了确定siRNA介导的mRNA抑制机制，我提出了一种双管齐下的方法：(1)深入分析siRNA靶mRNA在体内翻译和降解过程中依赖siRNA的变化；(2)Ago-siRNA复合物及其相互作用蛋白伙伴的生化纯化和表征。我将利用高通量测序技术来测试秀丽隐杆线虫和智人中sirna依赖的翻译和降解效应的几种不同模型，并利用秀丽隐杆线虫中丰富的功能遗传信息来确定任何观察到的效应所需的细胞机制。为了补充这种方法，我将直接识别与mRNA抑制相关的复合物，并评估它们对mRNA抑制的贡献，再次利用秀丽隐杆线虫的遗传资源。这些方法将为mRNA抑制的siRNA依赖机制提供更详细和全面的视角，所采用的技术将适用于许多系统中的siRNA研究。最终，了解sirna的分子效应和机制对于充分利用RNAi的临床和研究潜力，理解脱靶效应的生物学基础以及进一步了解小RNA生物学是必要的。

DESCRIPTION (provided by applicant): Short interfering RNAs (siRNAs) are small RNA regulators capable of reducing protein expression of their target mRNAs in a process known as RNA interference (RNAi). RNAi is important for normal cellular physiology and adaptation, as well as an invaluable genetic tool for scientists and a promising source of clinical therapeutics. While canonical models of RNAi feature siRNA-directed target mRNA cleavage by Argonaute proteins (Agos), recent studies have demonstrated that Ago-mediated cleavage is dispensable for target mRNA repression. Additional studies and comparisons with micro RNAs suggest that siRNAs may repress their target mRNAs via Ago-dependent translational repression and/or stimulation of degradation through non-Ago nucleases. To determine the mechanism(s) of siRNA-mediated mRNA repression, I propose a two-pronged approach: (1) a thorough analysis of siRNA-dependent changes in translation and degradation for siRNA target mRNAs in vivo and (2) biochemical purification and characterization of Ago-siRNA complexes and their interacting protein partners. I will utilize high throughput sequencing technologies to test severa different models of siRNA-dependent effects on translation and degradation in both C. elegans and H. sapiens, and take advantage of the wealth of functional genetic information in C. elegans to determine the cellular machinery required for any observed effect(s). To complement this approach I will directly identify the complexes associated with mRNA repression and assess their contributions to mRNA repression, again exploiting C. elegans' genetic resources. These approaches will provide a more detailed and comprehensive view of siRNA- dependent mechanism(s) of mRNA repression, and the techniques employed will be applicable to siRNA studies in many systems. Ultimately, knowledge of siRNAs' molecular effectors and mechanism(s) will be necessary for harnessing RNAi's full clinical and research potential, understanding the biological basis of off- target effects, and furthering our understanding of small RNA biology.