Investigating the Ago Cleavage Independent Mechanism(s) of RNAi

研究 RNAi 的 Ago 切割独立机制

• 批准号: 9187825
• 负责人: Joshua Arribere
• 金额: $ 3.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187825
• 关键词: Adoption; Affect; Alleles; Animals; Basic Science; Binding; Biochemical; Biological; Biology; Caenorhabditis elegans; Cell physiology; Cells; Cleaved cell; Clinical; Clinical Research; Complement; Complex; Dependency; Dissection; Fire - disasters; Future; Gene Expression; Gene Expression Regulation; Gene Silencing; Genetic; Genetic Translation; German population; Goals; High-Throughput Nucleotide Sequencing; Hybrids; Knowledge; Link; Mediating; Messenger RNA; Metabolism; Methods; MicroRNAs; Modeling; Molecular; Monitor; Organism; Pathway interactions; Process; Production; Proteins; RNA; RNA Interference; Recruitment Activity; Repression; Research Personnel; Ribonuclease H; Ribonucleoproteins; Ribosomes; Role; Scientist; Small Interfering RNA; Small RNA; Source; System; Techniques; Technology; Testing; Therapeutic; Time; Translational Repression; Translations; Work; base; comparative; design; experimental study; flexibility; genetic information; genetic resource; in vivo; loss of function; mRNA Expression; novel; nuclease; protein expression; public health relevance; stem; success; tool

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.

描述（由申请人提供）：短干扰RNA（siRNA）是小RNA调节剂，能够在称为RNA干扰（RNAi）的过程中减少其靶mRNA的蛋白质表达。 RNAi 对于正常细胞生理学和适应非常重要，也是科学家的宝贵遗传工具和临床治疗的有前途的来源。虽然 RNAi 的经典模型以 Argonaute 蛋白 (Agos) 进行 siRNA 指导的靶 mRNA 切割为特征，但最近的研究表明，Ago 介导的切割对于靶 mRNA 抑制来说是可有可无的。其他研究和与 micro RNA 的比较表明，siRNA 可能通过 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 抑制机制，我提出了一种双管齐下的方法：(1) 彻底分析体内 siRNA 靶标 mRNA 翻译和降解过程中 siRNA 依赖性变化，以及 (2) 对 Ago-siRNA 复合物及其相互作用的蛋白伴侣进行生化纯化和表征。我将利用高通量测序技术来测试线虫和智人中 siRNA 依赖性翻译和降解效应的几种不同模型，并利用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 依赖性机制提供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，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、了解脱靶效应的生物学基础以及加深我们对小 RNA 生物学的理解是必要的。