Investigating the Ago Cleavage Independent Mechanism(s) of RNAi

研究 RNAi 的 Ago 切割独立机制

• 批准号: 8783263
• 负责人: Joshua Arribere
• 金额: $ 4.99万
• 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 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783263
• 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; Future; Gene Expression; Gene Expression Regulation; Genetic; Genetic Translation; Goals; High-Throughput Nucleotide Sequencing; 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; flexibility; genetic resource; in vivo; loss of function; mRNA Expression; novel; nuclease; protein expression; public health relevance; research study; 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）是小的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对于正常的细胞生理和适应很重要，也是科学家的宝贵遗传工具，也是有前途的临床治疗来源。虽然RNAi的规范模型通过Argonaute蛋白（AGO）（AGOS）进行了siRNA指导的靶标mRNA裂解，但最近的研究表明，对于靶mRNA抑制，AGO介导的裂解是可分配的。其他研究和与微RNA的比较表明，siRNA可以通过AGO依赖性的翻译抑制和/或通过非核酸核酸酶抑制其靶标mRNA。为了确定siRNA介导的mRNA抑制的机制，我提出了一种两种原始的方法：（1）对siRNA依赖性变化的siRNA依赖性变化和体内siRNA靶标mRNA的变化，以及（2）（2）AGO-siRNA复合物及其相互作用蛋白质伴侣的生物化学纯化和表征。我将利用高吞吐量测序技术来测试Severa对秀丽隐杆线虫和H. sapiens中siRNA依赖性影响的不同模型，并利用秀丽隐杆线虫中的大量功能性遗传信息来确定任何观察到的效果所需的细胞机械（S）。为了补充这种方法，我将直接确定与mRNA抑制相关的复合物，并评估它们对mRNA抑制的贡献，再次利用秀丽隐杆线虫的遗传资源。这些方法将提供对mRNA抑制的siRNA依赖机制的更详细，更全面的看法，并且所采用的技术将适用于许多系统中的siRNA研究。最终，对于利用RNAi的完整临床和研究潜力，了解非靶向效应的生物学基础，并进一步了解我们对小RNA生物学的理解，对siRNAS的分子效应子和机制的了解将是必要的。