Structural and Mechanistic basis for RNA Silencing

项目摘要/摘要 RNA沉默是对一组不同的生物过程的统称，在这些过程中，小RNA包括 Piwi相互作用RNA(PiRNAs)、microRNAs(MiRNAs)和小干扰RNAs(SiRNAs)沉默 基因。这项建议旨在利用我的团队在结构生物学和生物化学方面的专业知识来 解决阻碍当前理解RNA的悬而未决的问题 沉默的生物学。具体地说：1)piRNAs，对保护基因组免受转座的影响是必不可少的 元素(TES)，已经用分子遗传学进行了广泛的研究，但是piRNA是如何与 在动物身上观察到的强大的TE沉默是由多种因素共同作用产生的，目前尚不清楚。我们会 通过发现RNA-蛋白质的结构和生化活性来解决这一知识鸿沟 与piRNA功能有关的复合体。2)miRNAs是细胞基因表达的普遍调节因子 但是miRNAs本身是如何被调控的，直到最近才被曝光。我们将继续前进 这一新兴领域通过确定靶向泛素化和破坏miRNA的结构基础- 最近发现的ZSWIM8库林环E3泛素连接酶的蛋白质复合体。3)虽然 MiRNAs的典型功能是沉默靶向的mRNAs，某些RNA病毒已经进化 改变细胞miRNAs的用途以稳定其病毒基因组并刺激病毒的机制 制作。MiRNA复合体被重定向以实现这些非规范的机制 人们对其功能知之甚少。我们正在通过调查互动来解决这一知识鸿沟 丙型肝炎病毒(丙型肝炎病毒)和肝脏特异性miRNA miR-122之间的关系，重点是了解 调节丙型肝炎病毒翻译以实现丙型肝炎病毒复制的miR-122-丙型肝炎病毒RNA复合体的结构 核糖核酸。4)RNA沉默领域最古老和最耐人寻味的谜团之一是，在某些情况下， 沉默的RNA很容易在动物细胞之间传递。在这一发现25年后，RNA的机制 进入动物细胞的运输仍不清楚。我们正在通过确定 广泛保守的整体膜SID-1识别和转运RNA的结构基础 将双链RNA运输到动物细胞中的蛋白质。合并后的研究预计将 提出克服上述主要知识障碍所需的见解及其影响 了解人类健康、生育能力、病毒感染和基于RNA的疗法的进展。

Project Summary/Abstract RNA Silencing is a blanket term for a diverse group of biological processes in which small RNAs, including PIWI-interacting RNAs (piRNAs), microRNAs (miRNAs), and small interfering RNAs (siRNAs) silence genes. This proposal aims to leverage my group’s expertise in structural biology and biochemistry to address outstanding questions that stand as roadblocks to advancing the current understanding of RNA Silencing biology. Specifically: 1) piRNAs, essential for defending the genome against transposable elements (TEs), have been studied extensively using molecular genetics, but how piRNA-associated factors work together to produce the potent TE-silencing observed in animals remains unclear. We will address this knowledge gap by discovering the structures and biochemical activities of RNA-protein complexes central to piRNA function. 2) miRNAs are ubiquitous regulators of cellular gene expression in mammals, but how miRNAs themselves are regulated has only recently come to light. We will advance this emerging area by determining the structural basis for targeted ubiquitination and destruction of miRNA- protein complexes by the recently discovered ZSWIM8 Cullin-RING E3 ubiquitin ligase. 3) Although the canonical function of miRNAs is to silence targeted mRNAs, certain RNA viruses have evolved mechanisms to repurpose cellular miRNAs to instead stabilize their viral genomes and stimulate viral production. The mechanisms by which miRNA complexes are redirected to achieve these non-canonical functions are poorly understood. We are addressing this knowledge gap by investigating interactions between Hepatitis C Virus (HCV) and the liver-specific miRNA miR-122, with a focus on understanding the structures of miR-122–HCV RNA complexes that modulate HCV translation to enable replication of HCV RNA. 4) One of the oldest and most intriguing mysteries of the RNA Silencing field is that, in some cases, silencing RNAs readily pass between animal cells. 25 years after this discovery, the mechanisms for RNA transport into animal cells remain unclear. We are addressing this shortcoming by determining the structural basis for RNA recognition and transport by SID-1, a broadly conserved integral membrane protein that transports double-stranded RNA into animal cells. The combined studies are expected to produce insights necessary to overcome the major knowledge barriers described above, with implications for understanding human health, fertility, viral infection, and the advancement of RNA-based therapies.