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

项目摘要 微小RNA（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，Dicer测量了从3' 2- 当裁剪环区域时，核苷酸（nt）突出端。将得到的19~23-nt的miRNA双链体加载 Argonaute蛋白（AGO）。因此，miRNAs被定义为长度为19~23个核苷酸（nt）的大小。 大多数使用下一代RNA测序（RNAseq）的关于miRNA的早期研究排除了~18 nt 短RNA，因此对这种微小的RNA（tyRNA）知之甚少。然而，最近的研究报告说， 许多tyRNA实际上与AGO结合，尽管它们的生理规律仍然未知。长期 该项目的目标是全面了解tyRNA的生理作用，并确定它们的功能。 生物合成途径短期目标是集中在一种特定类型的tyRNA，能够转化 AGO 3到AGO 2这样的切片机。我们的初步研究发现了这种tyRNA，并将其命名为 诱导切割的tyRNA（cityRNA）。此外，我们还鉴定了一种核酸酶， 引导RNA至14 nt cityRNA，从而激活AGO 3进行RNA切割。在这项研究中，我们假设 几种核酸酶将AGO结合的miRNA缩短为tyRNA，其中一些作为cityRNA， 催化活性AGO 3。为了验证这一假设，我们将追求以下具体目标。在Aim中 1，使用不同的指导RNA和AGO 3突变体的切割测定将用于确定 cityRNA和AGO 3对靶切割的需求。我们还将确定的晶体结构， AGO 3与cityRNA复合，这将为cityRNA的识别提供结构基础， AGO 3.在目标2中，将使用使用不同靶标的切割测定来确定以下的要求： 靶向RNA用于被负载cityRNA的AGO 3切割。我们将解决复杂的AGO 3的晶体结构 与cityRNA及其靶RNA的相互作用，这将阐明靶识别的机制。在目标3中， RNAseq和转录组分析将确定内源性cityRNA和由cityRNA切割的靶标。 AGO 3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 cityRNA介导的RNA切割机制及其与天然免疫的关系 反应

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.