Target specificity of human RNA-induced silencing complex

人RNA诱导的沉默复合物的靶标特异性

• 批准号: 10522487
• 负责人: Kotaro Nakanishi
• 金额: $ 33.56万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 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/10522487
• 关键词: 5' Untranslated Regions; Acylation; Algorithms; Amino Acids; Antiviral Response; Binding; Binding Proteins; Binding Sites; Biological Assay; Biological Process; Cells; Chimera organism; Complex; Cryoelectron Microscopy; Crystallization; Designer Drugs; Development; Disease; Dissociation; Drug Design; Equipment and supply inventories; Eukaryotic Cell; Event; Foundations; Funding; Gene Expression; Gene Silencing; Goals; Guide RNA; HIV; Human; Hydroxyl Radical; Immunoprecipitation; Individual; Internal Ribosome Entry Site; Investigation; Knowledge; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; MicroRNAs; Molecular; Molecular Conformation; N-methylisatoic anhydride; Nucleotides; Outcome; Outcome Study; Primer Extension; Property; Proteins; RNA; RNA Binding; RNA Interference; RNA-Induced Silencing Complex; Resolution; Role; Shapes; Site; Solid; Specificity; Structure; Techniques; Testing; Therapeutic; Translational Repression; Translations; Work; X-Ray Crystallography; base; design; experimental study; flexibility; hydroxyl group; improved; insight; mutant; neurodevelopment; novel; novel therapeutic intervention; prediction algorithm; prevent; scaffold; transcriptome sequencing

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.

项目摘要 在真核细胞中，基因表达在多个水平上受到调节，包括转录后基因 沉默，其中microRNA（miRNA）与互补靶RNA结合并引起翻译抑制。 Argonaute（AGO）蛋白质和miRNA形成RNA诱导的沉默复合物（RISC），核心参与者 基因沉默。人类有四个以前的蛋白质，AGO1-4，具有高序列身份，并且 在所有AGO中，大多数miRNA结合都是常见的。因此，人们认为这四个AGO 冗余地工作。尽管如此，越来越多的研究发现，每个以前都有其独特的 除基因沉默外，在各种生物过程和疾病中的作用。虽然所有人的相互作用 四个带有miRNA的AGO的表征很好，对每个RISC如何识别其目标知之甚少 RNA。阐明这种识别将为每个以前的独特角色提供洞察力。同时， RISC和目标相互作用的表征将通过改善预测来促进目标预测准确性 算法，它将不仅要考虑指南和目标之间的互补性 以前和目标相互作用。在这项拟议的研究中，我们将追求以下特定目标。在AIM 1中，我们 将使用冷冻电子显微镜和X射线晶体学来确定这四个的结构 具有相同指南和目标RNA的同质纯化RISC，这将提供有关该的洞察力 四个AGO在目标识别方面的差异。在AIM 2中，为了澄清这些差异，我们最近开发了 一种基于形状的新技术，使我们能够可视化目标RNA结合的构象动力学 到Risc。该方法将使我们能够表征RISC绑定通道及其内部的这种相互作用 在单核苷酸分辨率下的外围，可以扩展以了解Riscs如何识别指南 - 埋在高度结构化靶RNA中的结合位点。在AIM 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的目标特异性，并将其功能引导到替代细胞事件。这 这项研究的结果将为超出基因沉默以外的领域提供坚实的基础，并使能够 在治疗应用中开发更高准确性的RNA药物设计的新策略。