Investigating the Ago Cleavage Independent Mechanism(s) of RNAi

描述(申请人提供)：短干扰RNAs(SiRNAs)是一种小RNA调节剂，能够在称为RNA干扰(RNAi)的过程中减少其目标mRNAs的蛋白质表达。RNAi对正常的细胞生理和适应很重要，对科学家来说也是一个宝贵的遗传工具，也是临床治疗的一个有前途的来源。虽然经典的RNAi模型以siRNA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。更多的研究和与microRNAs的比较表明，siRNAs可能通过依赖于AGO的翻译抑制和/或通过非AGO核酸酶刺激降解来抑制其靶mRNAs。为了确定siRNA介导的mRNA抑制的机制(S)，我提出了一个双管齐下的方法：(1)彻底分析依赖siRNA的体内siRNA靶mRNAs翻译和降解的变化；(2)AgO-siRNA复合体及其相互作用蛋白的生化纯化和鉴定。我将利用高通量测序技术在线虫和智人中测试几种不同模式的siRNA依赖对翻译和降解的影响，并利用线虫丰富的功能性遗传信息来确定任何观察到的效应所需的细胞机制(S)。为了补充这一方法，我将直接鉴定与信使核糖核酸抑制相关的复合体，并评估它们对信使信使抑制的贡献，再次利用线虫的遗传资源。这些方法将提供一个更详细和全面的视角，以了解依赖于小干扰RNA的机制(S)，所采用的技术将适用于许多系统的小干扰RNA的研究。最终，了解siRNAs的分子效应器和机制(S)将是必要的，以充分利用RNAi的临床和研究潜力，了解非靶标效应的生物学基础，并进一步了解小RNA生物学。

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.