HOLO RISC ASSEMBLY

HOLO RISC 组件

• 批准号: 8361130
• 负责人: ADAM K YUAN
• 金额: $ 1.23万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361130
• 关键词: Address; Biology; Complex; Cryoelectron Microscopy; Electron Microscopy; Funding; Gene Silencing; Goals; Grant; MicroRNAs; Molecular; Mouse-ear Cress; National Center for Research Resources; Principal Investigator; Protein Binding; Proteins; Protocols documentation; RNA Interference; RNA-Induced Silencing Complex; Research; Research Infrastructure; Resources; Science; Small RNA; Source; Structure; Technology; Testing; Thermus thermophilus; United States National Institutes of Health; base; cost; design; human disease; improved; in vivo; macromolecule; novel therapeutics; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ARGONAUTE1 protein is the key component of RNA Induced Silencing Complex (RISC), which is the catalytic protein machinery for small RNA guided gene silencing. Ago proteins bind to a variety of auxiliary protein factors in vivo to form holo-RISC to perform the function. Deficiency of these auxiliary factors significantly compromises RISC function. The overall goal is to perform structural analysis of holo-RISC in different status. Given the size of this holo-RISC (ranging from 1~2 MDa), electron cryo-microscopy turns out to be a preferred tool to reveal the structure of this complex. We will purify and identify miRNA holo-RISC from Arabidopsis thaliana and the analogous complex from Thermus thermophilus. We have monoclonal antibodie, which specifically recognize these holo-RISCs. We are testing protocols to obtain homogeneous holo-RISCs. We will address several fundamental and long standing questions in the field of RNA silencing: What factors constitute holo-RISC? How is holo-RISC assembled? Studies of the structure and assembly of holo-RISC will provide not only the structure by also the mechanistic understanding of RNAi. Such efforts will eventually facilitate rational design of new and improved gene-silencing technologies. Applications of RNA silencing technologies could have a significant impact on a broad spectrum of biomedical sciences, ranging from understanding the basic biology, uncovering the molecular basis of human disease, to developing novel therapeutics.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 ArGONAUTE1蛋白是RNA诱导沉默复合体(RISC)的关键组成部分，RISC是小RNA引导的基因沉默的催化蛋白质机制。在体内，AGO蛋白与多种辅助蛋白因子结合形成全息RISC来执行这一功能。这些辅助因素的缺乏显著影响了RISC的功能。总体目标是对不同状态下的全息结构进行结构分析。考虑到这种全息结构的大小(从1~2个丙二醛)，电子冷冻显微镜是揭示这种复合体结构的首选工具。 我们将从拟南芥中提纯并鉴定miRNA holo-RISC，并从Thermus thermophilus中分离鉴定类似的复合体。我们有能识别这些全息干细胞的单抗。我们正在测试获得同质全息干细胞的方案。我们将解决RNA沉默领域中的几个基本和长期存在的问题：什么因素构成全息RISC？HOLO-RISC是如何组装的？ 对全息RISC的结构和组装的研究不仅可以通过对RNAi机制的理解来提供结构信息，而且还可以从机制上理解RNAi。这些努力最终将促进新的和改进的基因沉默技术的合理设计。RNA沉默技术的应用可能会对广泛的生物医学科学产生重大影响，从理解基础生物学，揭示人类疾病的分子基础，到开发新的疗法。