Target specificity of human RNA-induced silencing complex

项目总结 在真核细胞中，基因表达受到多个水平的调控，包括转录后基因。 沉默，即microRNAs(MiRNAs)与互补的靶RNA结合，导致翻译抑制。 ArgAerte(AGO)蛋白质和miRNAs形成RNA诱导的沉默复合体(RISCs)，RISCs是 基因沉默。人类有四种AGO蛋白，Ago1-4，它们有很高的序列同源性，而 大多数与miRNAs结合的miRNA在所有AGO中都是常见的。因此，人们一直认为，四年前 多此一举。然而，越来越多的研究发现，每一年都有其独特的 除了基因沉默外，还在各种生物过程和疾病中发挥作用。尽管所有人之间的互动 四个具有miRNAs的AGO已经被很好地描述，对于每个RISC如何识别其目标知之甚少 RNA。对这一认识的阐明将使我们深入了解每一项以前的独特作用。同时， RISC和目标相互作用的表征将通过提高预测来促进目标预测的准确性 算法，不仅要考虑引导和目标之间的互补性，而且要考虑类型 和目标交互。在这项拟议的研究中，我们将追求以下具体目标。在目标1中，我们 将使用低温电子显微镜和X射线结晶学来确定这四个化合物的结构 使用相同的指南和靶RNA均质纯化的RISCs，这将为深入了解 四个AGO在目标识别方面的差异。在目标2中，为了澄清这些差异，我们最近开发了 一种新的基于形状的技术，它允许我们可视化目标RNA结合的构象动力学 致RISC。该方法将使我们能够表征RISC结合通道内的这种相互作用及其 并可扩展以了解RISCs是如何识别GUIDE- 埋藏在高度结构的靶RNA中的结合位点。在目标3中，我们将首先使用质谱学来识别 每个AGO的独特的蛋白质结合伙伴及其特定的相互作用部位。然后，我们将使用串联 免疫沉淀，然后进行RNA测序，以确定这些蛋白质的结合如何影响 每个AGO的靶标特异性，并将它们的功能导向可选的细胞事件。这个 这项研究的结果将为基因沉默以外的领域提供坚实的基础，并使 开发更高精度指南的新策略--治疗应用中的RNA药物设计。

PROJECT SUMMARY In eukaryotic cells, gene expression is regulated at multiple levels, including post-transcriptional gene silencing, where microRNAs (miRNAs) bind to complementary target RNAs and cause translational repression. Argonaute (AGO) proteins and miRNAs form RNA-induced silencing complexes (RISCs), the core players in gene silencing. Humans have four AGO proteins, AGO1-4, which share a high sequence identity, and the majority of miRNAs bound are common across all AGOs. Therefore, it has been thought that the four AGOs work redundantly. Nevertheless, an increasing number of studies have found that each AGO has its unique roles in various biological processes and diseases in addition to gene silencing. Although the interaction of all four AGOs with miRNAs has been well characterized, little is known about how each RISC recognizes its target RNAs. Elucidation of this recognition will provide insight into the unique roles of each AGO. Meanwhile, characterization of RISC and target interactions will facilitate target prediction accuracy by improving prediction algorithms, which will take account of not only the complementarity between guide and target but also the type of AGO and target interaction. In this proposed study, we will pursue the following specific aims. In Aim 1, we will use cryo-electron microscopy and X-ray crystallography to determine the structures of all four homogenously purified RISCs with the same guide and target RNAs, which will provide insight into the differences in target recognition by the four AGOs. In Aim 2, to clarify these differences, we recently developed a novel SHAPE-based technique which allows us to visualize the conformational dynamics of target RNA bound to RISC. The method will enable us to characterize this interaction within the RISC binding channel and its periphery at a single-nucleotide resolution and can be expanded to understand how RISCs recognize guide- binding sites buried within highly structured target RNAs. In Aim 3, we will first use mass spectrometry to identify the unique protein binding partners of each AGO and their specific sites of interaction. Then, we will use tandem immunoprecipitation, followed by RNA sequencing, to determine how the binding of these proteins influences the target specificity of each AGO and directs their functionality towards alternative cellular events. The outcome from this study will provide a solid foundation for fields beyond gene silencing and enable the development of new strategies for higher accuracy guide-RNA drug design in therapeutic applications.