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: 9368173
• 负责人: Kotaro Nakanishi
• 金额: $ 29.87万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9368173
• 关键词: Alternative Splicing; Base Pairing; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Calcium Channel; Cells; Cerebellar Ataxia; Characteristics; Chemicals; Collaborations; Complex; Crystallization; Development; Disease; Dissociation; Elements; Eukaryotic Cell; Gene Expression; Gene Silencing; Genetic Transcription; Goals; Guide RNA; Human; Internal Ribosome Entry Site; Length; Lobe; Masks; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; N-terminal; Nucleic Acid Binding; Nucleic Acids; Outcome Study; Parents; Pattern; Physiological; Play; Protein C; Proteins; RNA; RNA Binding; RNA Interference; RNA analysis; RNA-Binding Proteins; RNA-Induced Silencing Complex; Regulation; Ribonucleoproteins; Roentgen Rays; Role; Saccharomycetales; Shapes; Specificity; Structure; Testing; Transcript; Transcription Initiation; Translational Repression; Translations; Untranslated RNA; Variant; X-Ray Crystallography; base; bioprocess; chromatin remodeling; design; experimental study; innovation; mutant; nervous system disorder; novel therapeutics; prevent; scaffold; voltage

PROJECT SUMMARY In eukaryotic cells, gene expression is controlled at multiple different layers. One of them is post- transcriptional gene silencing where microRNAs (miRNAs) bind target mRNAs in a sequence complementary manner and cause translational repression and/or deadenylation. In humans, miRNAs are loaded onto one of four Argonaute proteins (AGOs), forming a ribonucleoprotein complex terms `RNA-induced silencing complex (RISC)'. The target specificity of the RISC has been defined solely by the base complementarity between the miRNA (guide) and target strands. The loaded guide strand occupies part of the nucleic acid-binding channel between the AGO N-terminal and C-terminal lobes, while the remaining space serves as the composite target-binding channel. In this study, we hypothesize that the target specificity of the RISC is defined by the structure of the composite channel rather than just base complementarity, and thus that four human AGOs possess different target specificities due to their unique local structures. To validate this hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assay and chemical probing will be used to determine how differently target strands are recognized in the presence and absence of the N- terminal lobe. In Aim 2, X-ray crystallography will be used to solve the structure of human AGO3-RISC. This structure, along with the previously determined ones, will enable us to identify local structures making their target-binding channels different from each other. RNA bind-n-seq experiments using wild type and its mutant lacking the identified unique local structure(s) will determine the target specificities conferred by the characteristic target-binding channel. In Aim 3, filter-binding assays and chemical probing will be used to elucidate the molecular mechanism by which a miRNA, miR-3191-5p, activates only AGO4 for binding to the internal ribosome entry site (IRES) of CACNA1A mRNA, and blocks its IRES-driven translation to prevent the neurological disease. Outcomes from this study will provide a new concept on the target specificity of the RISC, which is significant because beyond canonical gene silencing, many different cellular bioprocesses are regulated by miRNAs.

项目摘要 在真核细胞中，基因表达在多个不同的层中受到控制。其中之一是 转录基因沉默，其中microRNA（miRNA）以序列互补结合靶标mRNA 方式并引起翻译抑制和/或去甲基化。在人类中，miRNA被加载到一个 四种Argonaute蛋白（AGO），形成核糖核蛋白复合物术语`RNA诱导的沉默复合物 （RISC）'。 RISC的目标特异性仅由基本互补性来定义 miRNA（指南）和目标链。加载的导链占据了核酸结合的一部分 AGO N末端和C末端裂片之间的通道，其余空间用作 复合目标结合通道。在这项研究中，我们假设RISC的目标特异性是 由复合通道的结构定义，而不仅仅是基础互补性，从而四个 由于其独特的局部结构，人类AGO具有不同的目标特异性。验证这一点 假设，我们将追求以下特定目标。在AIM 1中，切割测定和化学探测将是 用于确定在存在和不存在N-的情况下识别目标链的不同 终端叶。在AIM 2中，X射线晶体学将用于解决人类AGO3-RISC的结构。这 结构以及先前确定的结构将使我们能够确定局部结构 目标结合通道彼此不同。 RNA结合-N-Seq实验使用野生型及其 缺乏确定的独特局部结构的突变体将确定赋予的目标特异性 特征目标结合通道。在AIM 3中，将使用过滤器结合测定和化学探测 阐明miRNA miRNA，miR-3191-5p仅激活AGO4的分子机制，以与 CACNA1A mRNA的内部核糖体进入位点（IRES），并阻止其IRES驱动的翻译以防止 神经疾病。这项研究的结果将提供有关目标特异性的新概念 RISC很重要，因为超出规范基因沉默之外，许多不同的细胞生物处理是 由miRNA调节。