Target specificity of human RNA-induced silencing complex

项目摘要 在真核细胞中，基因表达在多个不同的层面上受到控制。其中一个是后- 转录基因沉默，其中微小RNA（miRNAs）以互补序列结合靶mRNA 方式并引起翻译抑制和/或去腺苷化。在人类中，miRNAs被加载到 四个Argonaute蛋白（AGO），形成一个核糖核蛋白复合体术语“RNA诱导沉默复合体 （RISC）RISC的靶特异性仅由RISC之间的碱基互补性定义。 miRNA（引导）和靶链。加载的引导链占据核酸结合链的一部分。 AGO N-末端和C-末端叶之间的通道，而剩余的空间用作 复合目标结合通道。在这项研究中，我们假设RISC的靶特异性是 由复合通道的结构定义，而不仅仅是碱基互补，因此， 人类AGO由于其独特的局部结构而具有不同的靶特异性。验证此 假设，我们将追求以下具体目标。在目标1中，将进行切割测定和化学探测。 用于确定在存在和不存在N-末端的情况下，靶链的识别方式如何不同。 顶叶在目标2中，X射线晶体学将用于解决人AGO 3-RISC的结构。这 结构，沿着与以前确定的，将使我们能够确定当地的结构，使他们的 目标结合通道彼此不同。使用野生型及其突变体的RNA结合-n-seq实验 缺乏所鉴定的独特局部结构的突变体将决定由所述突变体赋予的靶特异性。 特征目标结合通道。在目标3中，将使用滤膜结合试验和化学探测， 阐明了一种miRNA，miR-3191- 5 p，仅激活AGO 4与细胞结合的分子机制。 CACNA 1A mRNA的内部核糖体进入位点（IRES），并阻断其IRES驱动的翻译，以防止CACNA 1A mRNA的翻译。 神经系统疾病本研究的结果将提供一个新的概念的目标特异性的 RISC，这是重要的，因为除了典型的基因沉默，许多不同的细胞生物过程， 由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.