Target specificity of human RNA-induced silencing complex

家长奖项目摘要/摘要 在人类中，microRNAs(MiRNAs)被装载到四种ArgAerte(AGO)蛋白中，形成RNA诱导的沉默 复合体(RISC)，靶向基因沉默的mRNAs。这四种氨基酸的含量约为80% 身份和他们绑定的miRNA套件中甚至75%的miRNA激发了这样的想法，即四个AGO针对的是相同的一组 冗余的RNAs。然而，除了RNA干扰之外，每一个AGO都在神经中起着特殊的作用 发展、抗病毒反应和癌症。这一领域的一个明显的知识空白是 各具特色的功能各不相同。这一空白是由于对目标识别的不完全理解造成的。 每一件事的决定因素。到目前为止，所有的导引结合位点预测算法都只考虑了导引目标 互补性，但不是装载指南的类型。我们将检验这一假设，即每一次之前 细微差别的结构特征和互动的合作伙伴塑造了四个AGO-RISC每一个的目标清单 复合体。我们的长期目标是建立一种算法，能够通过使用 说明了人类农业的独特属性。为了实现这一总体目标，我们将开展以下工作 三个具体目标。在目标1中，我们将确定所有四个人类的冷冻电子显微镜和晶体结构。 AGOS绑定到相同的指南和目标RNA。我们已经纯化了均一的Ago1-，AgO2-和 AgO_3-RISCs(即，miR-20a-负载前)，并获得结合到 一个25个核苷酸的靶向RNA。我们还得到了这些配合物的初步晶体。我们将使用过滤器绑定 测定不同均一的AgO-RISC复合体结合的靶RNA的比例。结果是 来自目标1将为四个人类群体的区分目标识别提供结构基础。在目标2中， 我们将使用引物延伸分析的选择性2‘羟基酰化(SHAPE)来阐明这四个 人类AGOS识别靶RNA上的引导结合位点和侧翼区域。要了解RISCs如何 识别埋藏在高结构靶RNA中的引导结合位点，我们将用352- 人类免疫缺陷病毒5‘端非翻译区RNA和核糖体内部进入位点的176个核苷酸片段 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，我们将创造 并检测突变体与USP34和SART3的相互作用，以确定它们的结合部位 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小球进行免疫沉淀，然后进行RNA测序以识别结合的RNA。这 战略将应用于其他AGO，以确定其各自的特定合作伙伴清单和边界 RNA。AIM 3的结果将揭示AGOS的蛋白质伙伴如何对其靶标特异性做出贡献。 总之，拟议的项目将为领域提供坚实的基础，并帮助新的治疗策略 设计一类新的引导RNA药物，为每个人量身定做。

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.