Investigating the Ago Cleavage Independent Mechanism(s) of RNAi

描述（由申请人提供）：短干扰RNA（siRNA）是能够在称为RNA干扰（RNAi）的过程中降低其靶mRNA的蛋白质表达的小RNA调节剂。RNAi对于正常的细胞生理和适应非常重要，也是科学家的宝贵遗传工具和有前途的临床治疗来源。虽然RNAi的典型模型以Argonaute蛋白（Agos）介导的siRNA靶mRNA切割为特征，但最近的研究表明，Ago介导的切割与靶mRNA抑制无关。其他研究和与微小RNA的比较表明，siRNA可以通过Ago依赖性翻译抑制和/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抑制的机制，我提出了一个双管齐下的方法：（1）彻底分析siRNA靶mRNA在体内翻译和降解的siRNA依赖性变化和（2）Ago-siRNA复合物及其相互作用蛋白伴侣的生化纯化和表征。我将利用高通量测序技术来测试几种不同的siRNA依赖性对两种C. elegans和H. sapiens中的功能遗传信息，并利用C. elegans来确定任何观察到的效果所需的细胞机制。为了补充这种方法，我将直接鉴定与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C。线虫的遗传资源这些方法将提供mRNA抑制的siRNA依赖性机制的更详细和全面的视图，并且所采用的技术将适用于许多系统中的siRNA研究。最终，siRNA的分子效应子和机制的知识对于利用RNAi的全部临床和研究潜力、理解脱靶效应的生物学基础以及促进我们对小RNA生物学的理解将是必要的。

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.