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: 10647680
• 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/10647680
• 关键词: 3' Flanking Region; Binding; Binding Proteins; Biogenesis; Biological Assay; Complex; Data; Development; Exclusion; Exonuclease; Gene Expression; Genes; Goals; Guide RNA; Hela Cells; Human; In Vitro; Innate Immune Response; Interferons; Length; Measures; Methods; MicroRNAs; Molecular; Names; Natural Immunity; Neurodevelopmental Disorder; Nucleotides; Outcome; Outcome Study; Pathway interactions; Phosphodiesterase I; Physiological; Positioning Attribute; Proteins; RBM6 gene; RNA; RNA Interference; RNA-Induced Silencing Complex; Reporting; Repression; Role; Site; Slice; Structure; Testing; Untranslated RNA; Variant; Virus Diseases; Work; immune activation; 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.

项目概要 MicroRNA (miRNA) 是调节真核物种基因表达的非编码 RNA。 它们的前体具有茎环结构，Dicer 测量距 3'2- 剪切环区域时核苷酸 (nt) 突出。加载所得的 19~23-nt miRNA 双链体 转化为 Argonaute 蛋白 (AGO)。因此，miRNA的大小被定义为19~23个核苷酸(nt)长度。 大多数使用下一代 RNA 测序 (RNAseq) 进行的 miRNA 早期研究排除了约 18 nt 短RNA，因此人们对这种微小RNA（tyRNA）知之甚少。然而，最近的研究报道 许多 tyRNA 实际上与 AGO 结合，尽管它们的生理规则仍然未知。长期来看 该项目的目标是全面了解 tyRNA 的生理作用并确定其作用 生物发生途径。短期目标是专注于能够转化为特定类型的 tyRNA AGO3 到类似 AGO2 的切片机。我们的初步研究发现了这样的tyRNA，并将其命名为 “切割诱导 tyRNA (cityRNA)。”此外，我们还鉴定了一种核酸酶，可以修剪 AGO3 结合的核酸酶 将 RNA 引导至 14 nt cityRNA，从而激活 AGO3 进行 RNA 切割。在这项研究中，我们假设 一些核酸酶将 AGO 结合的 miRNA 缩短为 tyRNA，其中一些作为 cityRNA 发挥作用， 催化激活AGO3。为了验证这一假设，我们将追求以下具体目标。瞄准 1、使用不同指导RNA和AGO3突变体的裂解测定将用于确定 cityRNA 和 AGO3 对靶标切割的要求。我们还将确定晶体结构 AGO3与cityRNA复合，将为cityRNA的识别提供结构基础 AGO3.在目标 2 中，将使用不同靶标的裂解测定来确定以下要求： 用于被负载 cityRNA 的 AGO3 切割的目标 RNA。我们将解决AGO3复杂的晶体结构 与cityRNA及其目标RNA一起，这将阐明目标识别的机制。在目标 3 中， RNAseq 和转录组分析将确定内源 cityRNA 和由 AGO3 在先天免疫反应中的作用。总而言之，这项研究的结果将揭示分子 cityRNA介导的RNA切割机制以及cityRNA与先天免疫的相关性 回复。

项目成果

Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs.

• DOI: 10.1038/s41598-023-46562-6
• 发表时间: 2023-11-13
• 期刊: Scientific reports
• 影响因子: 4.6

Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA.

• DOI: 10.1073/pnas.2015026117
• 发表时间: 2020-11-17
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Park MS;Sim G;Kehling AC;Nakanishi K
• 通讯作者: Nakanishi K

Anatomy of four human Argonaute proteins.

• DOI: 10.1093/nar/gkac519
• 发表时间: 2022-07-08
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Nakanishi, Kotaro

Are Argonaute-Associated Tiny RNAs Junk, Inferior miRNAs, or a New Type of Functional RNAs?

• DOI: 10.3389/fmolb.2021.795356
• 发表时间: 2021
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5
• 作者: Nakanishi K

Manganese-dependent microRNA trimming by 3'→5' exonucleases generates 14-nucleotide or shorter tiny RNAs.

• DOI: 10.1073/pnas.2214335119
• 发表时间: 2022-12-20
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Sim, GeunYoung;Kehling, Audrey C.;Park, Mi Seul;Secor, Jackson;Divoky, Cameron;Zhang, Huaqun;Malhotra, Nipun;Bhagdikar, Divyaa;El-Wahab, Ekram W. Abd;Nakanishi, Kotaro
• 通讯作者: Nakanishi, Kotaro