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

家长奖项目摘要/摘要 MicroRNAs(MiRNAs)是19~23个核苷酸(NT)长度的非编码RNA，装载到ArgAerte蛋白质中 (AGOS)组装RNA诱导的沉默复合体(RISCs)。在四个人类物种中，只有AGO2 当靶RNA与指南完全互补时，显示出具有切片器活性。我们最近发现， Ag3在装载其3‘8~9的miR-20a的14个核苷酸的微小RNA(TyRNAs)时成为AGO2的竞争切片器 NT被删除。相反，AGO2在加载这些tyRNAs时显著降低了切片活性。我们的 结果表明，AgO2和Ag03具有不同的最适片段长度的引导RNA的切片活性。我们 将这种tyRNAs定义为切割诱导tyRNAs(CiyRNAs)。我们最近鉴定了几种3‘→5’核酸外切酶 干扰素刺激的核酸外切酶基因20(ISG20)的体外表达和鉴定 至14个核苷酸，从而激活RISC进行RNA切割。尽管先前的研究报告病毒感染 提高ISG20的水平，人们对ISG20的作用知之甚少。我们假设，诱导的ISG20在之前- 结合引导RNA，从而将主切片器从AGO2切换到AG03，以响应病毒感染。在AIM 1，我们将替换City RNAs之间保守的核苷酸，并验证它们对靶切割的影响。至 研究Ag3特定的局部结构在识别City RNAs中的参与，我们将使Ag3突变 缺乏任何一个独特的基序，并测试它们的体外靶标切割。我们还将确定水晶 AGO_3与14个核苷酸的城市RNA的复合体结构，以了解AGO_3如何识别城市RNA。结果是 来自Aim1的报告将首次深入了解City RNA负载AgO3对催化激活的要求。在AIM 2，我们将阐明AgO_3的活化机制。确定序列的互补性 CityRNA和靶RNA是靶向切割所需的，我们将系统地结合单个核苷酸 14-NT miR-20a和let-7a的每个位置都不匹配。加载任一City RNAs后，AG3将 进行了体外切片活性测试。我们将制作靶RNA变体，并确定靶RNA的最小长度 城市RNA定向的RNA裂解所必需的。最后，我们将确定三元络合物的晶体结构 Ag3与14-ntmiR-20a或let-7a及其靶RNA的结合。来自AIM2的结果将提供分子 CityRNA负载的AgO_3切割靶标的基础。在目标3中，我们将在HeLa中表达FLAG-AGO2或FLAG-AG03 使用和不使用干扰素处理的细胞。然后，从免疫纯化的旗帜中提取RNA-AGO， 然后进行RNA测序。使用一种结合计算和方法的方法，我们将确定 HeLa细胞天然免疫激活过程中被AgO_3结合的CITY RNA切割的靶标。Aim3的成果 将确定干扰素诱导的城市RNA及其目标RNA。总之，拟议的项目将重新定义 MiRNAs取决于大小，并揭示了City RNAs沉默的另一层RNA。

Summary/Abstract of Parent Award Project MicroRNAs (miRNAs) are 19~23 nucleotide(nt)-length noncoding RNAs that are loaded into Argonaute proteins (AGOs) to assemble the RNA-induced silencing complexes (RISCs). Among four human AGOs, only AGO2 has been shown to have slicer activity when target RNAs are fully complementary to the guide. We recently discovered that AGO3 becomes a competitive slicer of AGO2 when loaded with 14-nt tiny RNAs (tyRNAs) of miR-20a whose 3’ 8~9 nt are deleted. In contrast, AGO2 drastically decreased the slicing activity when loaded with those tyRNAs. Our findings demonstrate that AGO2 and AGO3 have different optimum lengths of guide RNA for slicing activity. We defined such tyRNAs as cleavage-inducing tyRNAs (cityRNAs). We recently identified several 3’→5’ exonucleases in vitro and identified interferon-stimulated exonuclease gene 20 (ISG20) that can trim AGO3-bound 23-nt miR-20a to 14 nt and thereby activate the RISC for RNA cleavage. Although previous studies reported that viral infection increases the ISG20 level, little is known about the role of ISG20. We hypothesize that the induced ISG20 trims AGO- bound guide RNAs, thereby switching the main slicer from AGO2 to AGO3, to respond to the viral infection. In Aim 1, we will replace the nucleotides conserved among cityRNAs and validate their effect on target cleavage. To examine the involvement of AGO3-specific local structures in recognition of cityRNAs, we will make AGO3 mutants lacking either of the unique motifs and test them for in vitro target cleavage. We also will determine the crystal structures of AGO3 in complex with a 14-nt cityRNA to understand how AGO3 recognizes cityRNAs. The outcomes from Aim1 will provide the first insight into the requirements of cityRNA-loaded AGO3 for catalytic activation. In Aim 2, we will elucidate the mechanism of AGO3 activation. To determine the sequence complementarity between cityRNA and target RNAs required for target cleavage, we will systematically incorporate a single nucleotide mismatch at every position of the 14-nt miR-20a and let-7a. After loading with either of the cityRNAs, AGO3 will be tested for in vitro slicing activity. We will make target RNA variants and determine the minimum length of target RNA required for cityRNA-directed RNA cleavage. Lastly, we will determine the ternary complex crystal structures of AGO3 with the 14-nt miR-20a or let-7a and their target RNA. The outcomes from Aim2 will provide the molecular basis for target cleavage by cityRNA-loaded AGO3. In Aim 3, we will express FLAG-AGO2 or FLAG-AGO3 in HeLa cells with and without interferon treatment. Then, RNAs will be extracted from the immunopurified FLAG-AGO, followed by RNA sequencing. Using an approach that combines computational and methods, we will determine targets cleaved by AGO3-bound cityRNAs during innate immune activation in HeLa cells. The outcomes from Aim3 will identify the interferon-induced cityRNAs and their target RNAs. Altogether, the proposed projects will redefine miRNAs depending on the size and shed light on another layer of RNA silencing by cityRNAs.