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: 10582158
• 负责人: Kotaro Nakanishi
• 金额: $ 20万
• 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/10582158
• 关键词: 3' Flanking Region; Applications Grants; Award; Binding Proteins; Complex; Computing Methodologies; Crystallization; Exonuclease; Genes; Goals; Guide RNA; Hela Cells; Hot Spot; Human; Immune; In Vitro; Innate Immune Response; Interferons; Length; Light; Methods; MicroRNAs; Microscope; Molecular; Natural Immunity; Nucleotides; Outcome; Parents; Phosphodiesterase I; Physiological; Positioning Attribute; Proteins; RBM6 gene; RNA; RNA Interference; RNA-Induced Silencing Complex; Reporting; Role; Seeds; Site; Slice; Structure; Testing; Untranslated RNA; Variant; Virus Diseases; immune activation; in vitro testing; insight; mutant; transcriptome sequencing

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.

摘要/家长奖项目的摘要 microRNA（miRNA）是19〜23核苷酸（NT） - 长度非编码RNA，它们被加载到Argonaute蛋白中 （AGO）组装RNA诱导的沉默复合物（RISC）。在四个人类AGO中，只有Ago2是 当目标RNA完全完整到指南时，显示为切片器活动。我们最近发现 Ago3成为AGO2的竞争切片机，当时MiR-20a的14 nt小RNA（Tyrnas）加载了3'8〜9 nt被删除。相反，当加载这些泰尔纳人时，AGO2大大降低了切片活性。我们的 研究结果表明，AGO2和AGO3具有不同的切片活性指南RNA的最佳长度。我们 定义了诸如触发裂解的泰尔纳斯（Cityrnas）之类的泰纳斯。我们最近确定了几个3'→5'外丝丝 在体外并鉴定出干扰素刺激的外切核酸酶基因20（ISG20），该基因可以修剪AGO3结合23-nt miR-20a 到14 NT，从而激活RISC进行RNA裂解。尽管以前的研究报告了病毒感染 增加ISG20水平，对ISG20的作用知之甚少。我们假设诱导的ISG20装饰以前 - 绑定的指南RNA，从而将主切片机从AGO2切换到AGO3，以应对病毒感染。目标 1，我们将取代Cityrnas之间构成的核苷酸，并验证其对目标裂解的影响。到 检查AGO3特定本地结构的参与以识别Cityrnas，我们将制作AGO3突变体 缺少任何独特的基序，并测试它们是否在体外靶裂解。我们还将确定晶体 AGO3的结构与14-NT Cityrna建立了复杂性，以了解AGO3如何认识CITYRNAS。结果 来自AIM1将首先了解载有AGO3的CityRNA对催化激活的要求。目标 2，我们将阐明AGO3激活的机制。确定序列互补性 靶裂解所需的CityRNA和目标RNA，我们将系统地合并一个核苷酸 在14-nt mir-20a和let-7a的每个位置都不匹配。加载了任何一个Cityrnas之后，Ago3将是 测试了体外切片活性。我们将制作目标RNA变体，并确定目标RNA的最小长度 CityRNA指导的RNA裂解所需。最后，我们将确定的三元复合物晶体结构 AGO3带有14-nt miR-20a或Let-7a及其靶RNA。 AIM2的结果将提供分子 载有Ago3的CityRNA的目标裂解的基础3。在AIM 3中，我们将在Hela中表达Flag-ago2或Flag-ago3 接受和没有干扰素治疗的细胞。然后，RNA将从免疫保护的Flag-ago中提取 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细胞中先天免疫激活期间，AGO3结合的CityRNA裂解的靶标。 AIM3的结果 将确定干扰素引起的CityRNA及其目标RNA。拟议的项目总共重新定义 miRNA取决于Cityrnas的另一层RNA沉默的大小和灯光。