Target specificity of human RNA-induced silencing complex

家长奖项目概要/摘要 在人类中，微小RNA（miRNAs）被加载到四种Argonaute（AGO）蛋白中以形成RNA诱导的沉默 复合物（RISC），其靶向mRNA用于基因沉默。发现四种AGO共享约80%的氨基酸， 同一性，甚至75%的他们的结合的miRNA套件启发了这样的概念，即四个AGO靶向同一组 RNA冗余。然而，除了RNA干扰，每个AGO都有助于在神经细胞中发挥专门的作用。 发展，抗病毒反应和癌症。该领域的一个明显的知识差距是 每个AGO的独特功能。这种空白是由于对目标识别的不完整理解 每个AGO的决定因素。迄今为止，所有的导向结合位点预测算法都只考虑导向靶 在一些实施方案中，所述抗体可以是互补性的，但不是其中加载了指导物的AGO的类型。我们将测试每个AGO的假设 对于这四个AGO-RISC，每一个都有一些微妙的结构特征和相互作用的伙伴，它们形成了目标清单 配合物我们的长期目标是建立一个算法，能够预测指导结合位点， 人类AGO的独特属性。为了实现这一总体目标，我们将努力实现以下目标 三个具体目标。在目标1中，我们将确定所有四个人的冷冻电子显微镜和晶体结构 AGO与相同的引导和靶RNA结合。我们已经纯化了均一的AGO 1-，AGO 2-， AGO3-RISC（即，miR-20a-负载的AGO），并获得结合至miR-20a的AGO1-RISC和AGO2-RISC的初始结构。 25-nt靶RNA。我们还获得了这些配合物的初步晶体。我们将使用过滤器绑定 测定以测量由不同的均质AGO-RISC复合物结合的靶RNA的分数。成果 Aim 1的结构将为四种人类AGO的差异靶标识别提供结构基础。在目标2中， 我们将使用引物延伸（SHAPE）分析的选择性2 '羟基酰化来阐明四种中的每一种是如何 人AGO识别靶RNA上的向导结合位点和侧翼区。要了解RISC如何 识别埋藏在高度结构化的靶RNA中的向导结合位点，我们将用352- 人免疫缺陷病毒5 '非翻译区RNA和核糖体内进入位点176-nt片段 核糖核酸目标2的结果将量化四个AGO的动态目标识别。在目标3中，我们将创建 AGO1-AGO2嵌合体，并测试突变体与USP 34和SART 3的相互作用，以鉴定其结合位点 仅位于AGO1。然后，我们将FLAG-AGO1与Myc-USP 40或-SART 3共表达，并进行串联表达。 使用抗FLAG和抗Myc珠进行免疫沉淀，然后进行RNA测序以鉴定结合的RNA。这 战略将适用于其他AGO，以确定其各自的特定合作伙伴和约束清单 RNA。目标3的结果将揭示AGO的蛋白质伴侣如何有助于其靶标特异性。 总而言之，拟议的项目将提供一个坚实的基础，领域和援助新的治疗策略， 为每个AGO定制一类新的指导RNA药物。

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.