Structural and molecular basis for cityRNA(cleavage-inducing tiny RNA)-directed RNA cleavage by AGO3

AGO3 指导的 cityRNA（切割诱导微小 RNA）切割 RNA 的结构和分子基础

• 批准号: 10213789
• 负责人: Kotaro Nakanishi
• 金额: $ 29.86万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213789
• 关键词: 3' Flanking Region; Binding; Binding Proteins; Biogenesis; Biological Assay; Cleaved cell; Complex; Crystallization; Data; Development; Disease; Exonuclease; Gene Expression; Genes; Goals; Guide RNA; Hela Cells; Human; Immune; In Vitro; Innate Immune Response; Interferons; Length; Light; Measures; Methods; MicroRNAs; Molecular; Names; Natural Immunity; Nucleotides; Outcome; Outcome Study; Pathway interactions; Phosphodiesterase I; Physiological; Positioning Attribute; Proteins; RBM6 gene; RNA; RNA Interference; RNA-Induced Silencing Complex; Reporting; Role; Seeds; Site; Slice; Structure; Testing; Untranslated RNA; Variant; Virus Diseases; Work; base; in vitro testing; insight; mutant; next generation; nuclease; stem; transcriptome; transcriptome sequencing

PROJECT SUMMARY MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression in eukaryotic species. Their precursors have a stem-loop structure, and Dicer measures the distance from the 3' 2- nucleotide (nt) overhang when cropping the loop region. The resultant 19~23-nt miRNA duplexes are loaded into Argonaute proteins (AGOs). Therefore, miRNAs are defined by the size of 19~23 nucleotide (nt) length. Most of the early studies about miRNAs using next-generation RNA sequencing (RNAseq) excluded ~18 nt short RNAs, and thus little is known about such tiny RNAs (tyRNAs). However, recent studies reported that many tyRNAs actually bind to AGOs, although their physiological rule remains unknown. The long-term goal of this project is to understand the physiological role of tyRNAs comprehensively and to determine their biogenesis pathways. The short-term objective is to focus on a specific type of tyRNAs capable of converting AGO3 to a slicer like AGO2. Such tyRNAs were discovered by our preliminary studies and named `cleavage-inducing tyRNAs (cityRNAs).' In addition, we also identified a nuclease that trims AGO3-bound guide RNA to a 14 nt cityRNA, thereby activating AGO3 for RNA cleavage. In this study, we hypothesize that several nucleases shorten AGO-bound miRNAs to tyRNAs, some of which work as cityRNAs to catalytically activate AGO3. To validate this hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assays using different guide RNAs and AGO3 mutants will be used to determine the requirements of cityRNA and AGO3 for target cleavage. We will also determine the crystal structures of AGO3 in complex with cityRNAs, which will provide the structural basis for the recognition of cityRNAs by AGO3. In Aim 2, cleavage assays using different targets will be used to determine the requirements of target RNAs for cleavage by cityRNA-loaded AGO3. We will solve the crystal structures of AGO3 in complex with cityRNAs and their target RNA, which will elucidate the mechanism of the target recognition. In Aim 3, RNAseq and transcriptome analyses will determine the endogenous cityRNAs and targets cleaved by AGO3 in the innate immune response. Altogether, outcomes from this study will reveal the molecular mechanism of cityRNA-directed RNA cleavage and the correlation between cityRNAs and innate immune response.

项目摘要 microRNA（miRNA）是调节真核种类基因表达的非编码RNA。 它们的前体具有茎环结构，迪切尔测量了与3'2--的距离 裁剪循环区域时的核苷酸（NT）悬垂。最终的19〜23-nt miRNA双链体加载 进入Argonaute蛋白（AGO）。因此，miRNA由19〜23核苷酸（NT）长度的大小定义。 使用下一代RNA测序（RNASEQ）的大多数关于miRNA的早期研究不包括〜18 nt 简短的RNA，因此对这种微小的RNA（Tyrnas）知之甚少。但是，最近的研究报道了 尽管其生理规则仍然未知，但许多泰尔纳人实际上与AGO结合了。长期 该项目的目标是全面了解泰尔纳人的生理作用，并确定他们的生理作用 生物发生途径。短期目标是专注于能够转换的特定类型 AGO3到像Ago2这样的切片机。这样的泰尔纳人是通过我们的初步研究发现的，命名为 “裂解诱导的泰纳斯（Cityrnas）。”另外，我们还确定了一个核酸酶，该核酸酶修剪了AGO 3结合 将RNA引导至14 NT CityRNA，从而激活AGO3进行RNA裂解。在这项研究中，我们假设 几个核酸酶很短，以塔纳斯的束缚，其中一些是cityrnas的 催化激活AGO3。为了验证这一假设，我们将追求以下特定目标。目标 1，使用不同的指南RNA和AGO3突变体的分裂测定将用于确定 CityRNA和AGO3的要求对目标裂解的要求。我们还将确定 Ago3与Cityrnas的复杂性，这将为认可Cityrnas的结构基础 Ago3。在AIM 2中，使用不同目标的分裂测定将用于确定 target rnas cityrna载有Ago3的裂解。我们将在复合物中求解AGO3的晶体结构 使用CityRNA及其目标RNA，这将阐明目标识别的机制。在AIM 3中， RNASEQ和转录组分析将确定内源性城市NAS，并靶向裂开 AGO3在先天免疫反应中。总共，这项研究的结果将揭示分子 CityRNA指导的RNA裂解机制以及Cityrnas与先天免疫之间的相关性 回复。