Structural and Mechanistic basis for RNA Silencing

项目概要/摘要 RNA沉默是一组不同的生物过程的总称，其中小RNA，包括 PIWI 相互作用 RNA (piRNA)、microRNA (miRNA) 和小干扰 RNA (siRNA) 沉默 基因。该提案旨在利用我小组在结构生物学和生物化学方面的专业知识 解决阻碍当前对 RNA 理解的突出问题 沉默生物学。具体来说：1) piRNA，对于保护基因组免受转座至关重要 分子遗传学已对 piRNA 元素 (TE) 进行了广泛研究，但 piRNA 相关性如何 在动物中观察到的因素共同作用产生有效的 TE 沉默仍不清楚。我们会 通过发现 RNA-蛋白质的结构和生化活性来填补这一知识空白 piRNA 功能的核心复合物。 2) miRNA 是细胞基因表达的普遍调节因子 哺乳动物，但 miRNA 本身的调控方式直到最近才被揭示。我们将前进 通过确定 miRNA 靶向泛素化和破坏的结构基础来研究这一新兴领域 最近发现的 ZSWIM8 Cullin-RING E3 泛素连接酶产生的蛋白质复合物。 3）虽然 miRNA 的典型功能是沉默目标 mRNA，某些 RNA 病毒已经进化 重新利用细胞 miRNA 来稳定其病毒基因组并刺激病毒的机制 生产。 miRNA 复合体被重定向以实现这些非规范的机制 功能了解甚少。我们正在通过调查交互来解决这一知识差距 丙型肝炎病毒 (HCV) 和肝脏特异性 miRNA miR-122 之间的关系，重点是了解 调节 HCV 翻译以实现 HCV 复制的 miR-122-HCV RNA 复合物的结构 核糖核酸。 4) RNA 沉默领域最古老、最有趣的谜团之一是，在某些情况下， 沉默RNA很容易在动物细胞之间传递。这一发现 25 年后，RNA 的机制 转运到动物细胞中的情况仍不清楚。我们正在通过确定 SID-1（一种广泛保守的整合膜）识别和运输 RNA 的结构基础 将双链RNA转运到动物细胞中的蛋白质。综合研究预计将 产生克服上述主要知识障碍所需的见解，并产生影响 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.