Target specificity of human RNA-induced silencing complex

人RNA诱导的沉默复合物的靶标特异性

基本信息

批准号：
10797295
负责人：
Kotaro Nakanishi
金额：
$ 14.67万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10797295
关键词：
5&apos Untranslated Regions Acylation Algorithms Amino Acids Antiviral Response Applications Grants Award Binding Binding Sites Biological Assay Cells Chimera organism Complex Cryoelectron Microscopy Crystallization Designer Drugs Dissociation Equipment Malfunction Equipment and supply inventories Event Foundations Funding Gene Silencing Goals Guide RNA HIV Human Hydroxyl Radical Immunoprecipitation Individual Internal Ribosome Entry Site Investigation Knowledge Malignant Neoplasms Mass Spectrum Analysis Measures Messenger RNA Methods MicroRNAs Molecular N-methylisatoic anhydride Nucleotides Outcome Parents Primer Extension Property Proteins RNA RNA Binding RNA Interference RNA-Induced Silencing Complex Resolution Role Shapes Solid Specificity Structure Techniques Testing Visualization base design experimental study flexibility hydroxyl group imager mutant neurodevelopment novel therapeutic intervention prediction algorithm scaffold screening transcriptome sequencing

项目摘要

Summary/Abstract of Parent Award Project In humans, microRNAs (miRNAs) are loaded into four Argonaute (AGO) proteins to form RNA-induced silencing complexes (RISC), which target mRNAs for gene silencing. The finding that the four AGOs share ~80% amino acid identity and even 75% of their suite of bound miRNAs inspired the notion that the four AGOs target the same set of RNAs redundantly. However, in addition to RNA interference, each AGO contributes to specialized roles in neural development, antiviral response, and cancer. A clear knowledge gap in the field is the molecular bases for the distinctive functions of each AGO. This void is due to an incomplete understanding of the target-recognition determinants of each AGO. To date, all prediction algorithms of guide-binding sites only consider the guide-target complementarity but not the type of AGO in which the guide is loaded. We will test the hypothesis that each AGO's nuanced structural features and interacting partners shape the inventory of targets for each of the four AGO-RISC complexes. Our long-term goal is to establish an algorithm capable of predicting guide-binding sites by taking account of the unique properties of human AGOs. Toward this overarching objective, we will pursue the following three specific Aims. In Aim 1, we will determine the cryo-electron microscopy and crystal structures of all four human AGOs bound to the same guide and target RNAs. We have already purified homogeneous AGO1-, AGO2-, and AGO3-RISCs (i.e., miR-20a-loaded AGO) and obtained initial structures of AGO1-RISC and AGO2-RISC bound to a 25-nt target RNA. We have also obtained preliminary crystals for these complexes. We will use a filter-binding assay to measure the fraction of target RNAs bound by different homogeneous AGO-RISC complexes. The outcome from Aim 1 will provide the structural basis for the differential target recognition by the four human AGOs. In Aim 2, we will use Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to elucidate how each of the four human AGOs recognizes the guide-binding site and the flanking regions on target RNAs. To understand how RISCs recognize the guide-binding site buried in a highly structured target RNA, we will perform SHAPE studies with a 352- nt human immunodeficiency virus 5’ untranslated region RNA and a 176-nt fragment of ribosomal internal entry site RNA. The outcome from Aim 2 will quantify the dynamic target recognition of the four AGOs. In Aim 3, we will create AGO1-AGO2 chimeras and test the mutants for interaction with USP34 and SART3 to identify their binding site located only in AGO1. Then, we will co-express FLAG-AGO1 with Myc-USP40 or -SART3 and perform a tandem immunoprecipitation using anti-FLAG and anti-Myc beads, followed by RNA sequencing to identify bound RNAs. This strategy will be applied to the other AGOs to determine their respective inventory of specific partners and the bound RNAs. The outcome from Aim 3 will reveal how protein partners of AGOs contribute to their target specificity. Altogether, the proposed projects will provide a solid foundation for fields and aid new therapeutic strategies to design a new class of guide RNA drugs customized for each AGO.

摘要/家长奖项目的摘要在人类中，将microRNA（miRNA）加载到四个Argonaute（AGO）蛋白中以形成RNA诱导的沉默复合物（RISC），靶向基因沉默的mRNA。这四个AGO共享约80％氨基酸的发现身份，甚至75％的绑定miRNA套件都启发了四个AGO目标相同集的观念 rnas冗余。但是，除了RNA干扰外，每个AGO都在神经元中促进了专业角色发育，抗病毒反应和癌症。现场的明显知识差距是分子碱基每个前的独特功能。这个空白是由于对目标识别的不完全理解确定每个月。迄今为止，指南结合站点的所有预测算法仅考虑指南目标互补性，而不是加载指南的AGO类型。我们将测试每个前的假设细微的结构特征和互动合作伙伴塑造了四个AGO-RISC中每个目标的库存复合物。我们的长期目标是建立一种能够通过服用来预测指导站点的算法人类AGO的独特特性的描述。走向这个总体目标，我们将追求以下三个具体目标。在AIM 1中，我们将确定所有四个人类的低温 - 电子显微镜和晶体结构 AGO与同一指南和靶向RNA结合。我们已经纯化了Ago1-，Ago2-和 Ago3-Riscs（即MiR-20a负载AGO），并获得了AGO1-RISC和AGO2-RISC的初始结构与 25 nt靶RNA。我们还获得了这些复合物的初步晶体。我们将使用过滤器结合测量由不同均质AGO-RISC复合物约束的靶RNA的分数。结果来自AIM 1将为四个人类AGO的差异目标识别提供结构基础。在AIM 2中，我们将使用通过引物延伸（形状）分析的选择性2'羟基酰基化来阐明四个如何人类AGO认识到目标RNA的指南结合部位和侧翼区域。了解RISC如何认识到建立高度结构化的目标RNA的指导结合位点，我们将使用352--进行形状研究 NT人类免疫缺陷病毒5'未翻译区域RNA和176-nt核糖体内部入口部位 RNA。 AIM 2的结果将量化四个AGO的动态目标识别。在AIM 3中，我们将创建 AGO1-AGO2嵌合体并测试突变体与USP34和SART3的相互作用以识别其结合位点仅位于AGO1。然后，我们将与myc-usp40或-sart3共同表达flag-ago1并执行串联使用抗FLAG和抗MYC珠的免疫沉淀，然后进行RNA测序以鉴定结合的RNA。这策略将应用于其他AGO，以确定其各自的特定合作伙伴的清单和约束 RNA。 AIM 3的结果将揭示AGO的蛋白质伴侣如何促进其目标特异性。拟议的项目总共将为领域和帮助新的疗法策略提供坚实的基础设计一种新的指南RNA药物，每天都定制。