Target specificity of human RNA-induced silencing complex

项目总结 在真核细胞中，基因的表达在多个不同的层面上受到调控。其中一个是POST- 转录基因沉默，即microRNAs(MiRNAs)以互补序列与靶mRNAs结合 导致翻译抑制和/或去烯化。在人类中，miRNAs被加载到 四种精氨酸蛋白(AGO)，形成核糖核蛋白复合体，称为RNA诱导沉默复合体 (RISC)‘。RISC的靶特异性仅由RISC和RISC之间的碱基互补性定义 MiRNA(引导)和靶链。负载的导向链占据了部分核酸结合 在AGO N端和C端叶之间的通道，而剩余的空间用作 复合靶结合通道。在这项研究中，我们假设RISC的靶标特异性是 由复合通道的结构定义，而不仅仅是碱基互补，因此这四个 人类AGO由于其独特的局部结构而具有不同的靶标特异性。要验证此点，请执行以下操作 假设，我们将追求以下具体目标。在目标1中，将进行卵裂试验和化学探测 用于确定在存在和不存在N-的情况下识别靶链的不同程度 终叶。在目标2中，将使用X射线结晶学来解决人类AgO_3-RISC的结构问题。这 结构，以及之前确定的结构，将使我们能够识别本地结构，使其 靶结合通道彼此不同。利用野生型进行RNA Bind-n-Seq实验及其 缺乏已鉴定的唯一局部结构的突变体(S)将决定由 特有的靶向结合通道。在目标3中，将使用过滤器结合分析和化学探测来 阐明miRNA miR-3191-5P仅激活AGO4与 CACNA1AmRNA的内部核糖体进入位点(IRES)，并阻断其IRES驱动的翻译，以防止 神经系统疾病。这项研究的结果将提供一个关于目标特异性的新概念 RISC，这很重要，因为除了规范的基因沉默之外，许多不同的细胞生物过程 受miRNAs调控。

PROJECT SUMMARY In eukaryotic cells, gene expression is controlled at multiple different layers. One of them is post- transcriptional gene silencing where microRNAs (miRNAs) bind target mRNAs in a sequence complementary manner and cause translational repression and/or deadenylation. In humans, miRNAs are loaded onto one of four Argonaute proteins (AGOs), forming a ribonucleoprotein complex terms `RNA-induced silencing complex (RISC)'. The target specificity of the RISC has been defined solely by the base complementarity between the miRNA (guide) and target strands. The loaded guide strand occupies part of the nucleic acid-binding channel between the AGO N-terminal and C-terminal lobes, while the remaining space serves as the composite target-binding channel. In this study, we hypothesize that the target specificity of the RISC is defined by the structure of the composite channel rather than just base complementarity, and thus that four human AGOs possess different target specificities due to their unique local structures. To validate this hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assay and chemical probing will be used to determine how differently target strands are recognized in the presence and absence of the N- terminal lobe. In Aim 2, X-ray crystallography will be used to solve the structure of human AGO3-RISC. This structure, along with the previously determined ones, will enable us to identify local structures making their target-binding channels different from each other. RNA bind-n-seq experiments using wild type and its mutant lacking the identified unique local structure(s) will determine the target specificities conferred by the characteristic target-binding channel. In Aim 3, filter-binding assays and chemical probing will be used to elucidate the molecular mechanism by which a miRNA, miR-3191-5p, activates only AGO4 for binding to the internal ribosome entry site (IRES) of CACNA1A mRNA, and blocks its IRES-driven translation to prevent the neurological disease. Outcomes from this study will provide a new concept on the target specificity of the RISC, which is significant because beyond canonical gene silencing, many different cellular bioprocesses are regulated by miRNAs.