HOLO RISC ASSEMBLY

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。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 ARGONAUTE 1蛋白是RNA诱导沉默复合物（RISC）的关键组分，RISC是小RNA介导基因沉默的催化蛋白质机制。Ago蛋白在体内与多种辅助蛋白因子结合形成holo-RISC来执行功能。这些辅助因素的不足会严重影响RISC的功能。总体目标是对不同状态下的holo-RISC进行结构分析。考虑到该复合物的大小（1~2 MDa），冷冻电镜是揭示其结构的首选工具。 我们将纯化和鉴定来自拟南芥的miRNA holo-RISC和来自嗜热栖热菌的类似复合物。我们有单克隆抗体，可以特异性识别这些全RISC。我们正在测试获得同质全息RISC的协议。我们将解决RNA沉默领域的几个基本和长期存在的问题：什么因素构成了holo-RISC？Holo-RISC是如何组装的？ 对holo-RISC的结构和组装的研究将不仅提供RNAi的结构，还提供RNAi的机理理解。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对生物医学科学的广泛领域产生重大影响，从了解基础生物学，揭示人类疾病的分子基础到开发新的治疗方法。

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.