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

家长奖项目概要/摘要 微小RNA（microRNAs，miRNAs）是一种长度为19~23个核苷酸的非编码RNA，装载于Argonaute蛋白中 RNA诱导的沉默复合物（RISC）。在四种人类AGO中，只有AGO 2被发现。 当靶RNA与向导完全互补时，显示具有切片活性。我们最近发现 当AGO 3负载miR-20 a的14-nt微小RNA（tyRNA）时，AGO 3成为AGO 2的竞争性切割器， NT被删除。相比之下，AGO 2在装载有这些tyRNA时显著降低了切割活性。我们 结果表明，AGO 2和AGO 3具有不同的用于切割活性的指导RNA的最佳长度。我们 将这种tyRNA定义为裂解诱导tyRNA（cityRNA）。我们最近鉴定了几种3 '→ 5'核酸外切酶 体外和鉴定的干扰素刺激的外切核酸酶基因20（ISG 20），其可以修剪AGO 3结合的23-nt miR-20 a 至14 nt，从而激活RISC进行RNA切割。虽然先前的研究报告说，病毒感染 增加ISG 20水平，但对ISG 20的作用知之甚少。我们假设诱导的ISG 20修剪AGO- 结合向导RNA，从而将主要的切割器从AGO 2转换为AGO 3，以响应病毒感染。在Aim中 1，我们将替换cityRNA中保守的核苷酸，并验证它们对靶切割的影响。到 研究AGO 3特异性局部结构在识别cityRNA中的参与，我们将使AGO 3突变体 缺乏任何一种独特的基序，并测试它们的体外靶切割。我们还将确定水晶 AGO 3与14-nt城市RNA复合的结构，以了解AGO 3如何识别城市RNA。成果 来自Aim 1的研究将首次深入了解负载cityRNA的AGO 3催化活化的要求。在Aim中 2、阐明AGO_3的激活机制。确定之间的序列互补性 cityRNA和靶切割所需的靶RNA，我们将系统地掺入单个核苷酸 在14-nt miR-20 a和let-7a的每个位置处存在错配。在加载任何一种cityRNA后，AGO 3将被 测试体外切割活性。我们将制作靶RNA变体并确定靶RNA的最小长度 这是cityRNA定向RNA切割所必需的。最后，我们将确定三元复合物的晶体结构， AGO 3与14-nt miR-20 a或let-7a及其靶RNA的结合。Aim 2的结果将提供分子水平的 通过负载cityRNA的AGO 3进行靶切割的基础。在目标3中，我们将在HeLa中表达FLAG-AGO 2或FLAG-AGO 3 有和没有干扰素治疗的细胞。然后，从免疫纯化的FLAG-AGO中提取RNA， 然后进行RNA测序。使用结合计算和方法的方法，我们将确定 在HeLa细胞的先天免疫激活过程中被AGO 3结合的cityRNA切割的靶点。目标3的结果 将鉴定干扰素诱导的cityRNA及其靶RNA。总而言之，拟议的项目将重新定义 miRNAs的大小和揭示了cityRNAs的另一层RNA沉默。

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.