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

项目总结 MicroRNAs(MiRNAs)是真核生物中调控基因表达的非编码RNA。 它们的前体具有茎-环结构，迪特尔测量了从3‘2- 当修剪环状区域时，核苷酸(NT)突出。将得到的19~23-nT的miRNA双链加载 转化为ArgAerte蛋白质(AGOS)。因此，miRNAs被定义为19~23个核苷酸(NT)的长度。 大多数早期使用下一代RNA测序(RNAseq)的miRNAs研究排除了~18nt 短RNA，因此对这种微小的RNA(TyRNAs)知之甚少。然而，最近的研究报告说 许多tyRNA实际上与agos结合，尽管它们的生理规则尚不清楚。长期的 该项目的目标是全面了解tyRNAs的生理作用，并确定其 生物发生途径。短期目标是专注于一种特定类型的能够转化为 AG03到像AGO2这样的切片机。这种tyRNA是我们通过初步研究发现的，并命名为 “诱导分裂的tyRNAs(CiyRNAs)。”此外，我们还鉴定了一种核酸酶，它可以剪裁ag3结合的核酸酶。 引导RNA到14个核苷酸的城市RNA，从而激活Ag03进行RNA切割。在这项研究中，我们假设 几种核酸酶将以前结合的miRNAs缩短为tyRNAs，其中一些作为City RNAs作用于 催化激活AgO_3。为了验证这一假设，我们将追求以下具体目标。在AIM 1，使用不同的引导RNA和ag3突变体的切割分析将被用于确定 CityRNA和AgO_3对靶标切割的要求。我们还将确定其晶体结构。 AgO_3与City RNAs的络合物，这将为CityRNA的识别提供结构基础 AgO_3。在目标2中，将使用使用不同目标的切割分析来确定 靶向RNA以被装载城市RNA的Ag03切割。我们将在络合物中解决AgO_3的晶体结构 这将有助于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CITRNAs和被 AG03在先天免疫反应中的作用。总之，这项研究的结果将揭示分子 城市RNA介导的RNA切割机制及其与天然免疫的关系 回应。

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.