Target specificity of human RNA-induced silencing complex

项目摘要 在真核细胞中，基因表达在多个水平上受到调控，包括转录后基因调控。 沉默，其中微小RNA（miRNA）与互补靶RNA结合并引起翻译抑制。 Argonaute（AGO）蛋白和miRNAs形成RNA诱导沉默复合物（RISC），这是RNA诱导沉默复合物的核心参与者。 基因沉默人类有四种AGO蛋白，AGO 1 -4，它们具有高度的序列同一性， 大多数结合的miRNA在所有AGO中是共同的。因此，有人认为，这四个AGO 重复地工作。然而，越来越多的研究发现，每个AGO都有其独特的 除了基因沉默之外，它还在各种生物过程和疾病中发挥作用。虽然所有人的互动 四个含有miRNAs的AGO已经被很好地表征，但关于每个RISC如何识别其靶点却知之甚少 RNA。阐明这一认识将提供洞察每个AGO的独特作用。同时， RISC和靶相互作用的表征将通过提高预测精度来促进靶预测精度 算法，这将不仅考虑到指导和目标之间的互补性， AGO和目标相互作用。在这项拟议的研究中，我们将追求以下具体目标。目标1： 将使用低温电子显微镜和X射线晶体学来确定所有四个的结构 具有相同的引导和靶RNA的均质纯化的RISC，这将提供对 四个AGO的目标识别差异。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SHAPE的技术，使我们能够可视化靶RNA结合的构象动力学， 到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 外围在一个单核苷酸的分辨率，并可以扩展到了解RISC如何识别指导， 隐藏在高度结构化的靶RNA中的结合位点。在目标3中，我们将首先使用质谱法来识别 每个AGO的独特蛋白质结合伴侣及其相互作用的特定位点。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的靶特异性，并将其功能导向可选的细胞事件。的 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， 在治疗应用中开发更高准确性的指导RNA药物设计的新策略。

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.