Structural and Mechanistic basis for RNA Silencing

项目概要/摘要 RNA沉默是一个笼统的术语，用于描述一组不同的生物过程，其中小RNA，包括 PIWI相互作用RNA（piRNA）、microRNA（miRNAs）和小干扰RNA（siRNAs）沉默 基因.这项提案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， 解决阻碍当前对RNA理解的突出问题 生物学沉默。具体地说：1）piRNA，对于保护基因组免受转座所必需的 已经使用分子遗传学广泛地研究了piRNA相关的转录因子（TE），但是如何与piRNA相关的转录因子（TE）结合， 在动物中观察到的多种因素共同作用以产生有效的TE沉默仍然不清楚。我们将 通过发现RNA蛋白质的结构和生物化学活性来填补这一知识空白 复合物对皮尔纳功能至关重要。2)miRNA是细胞基因表达的普遍调节因子， 哺乳动物，但miRNAs本身是如何调节的，直到最近才被发现。我们将推进 通过确定靶向泛素化和破坏miRNA的结构基础， 蛋白质复合物通过最近发现的Cullin-RING E3泛素连接酶。3)虽然 miRNA的典型功能是沉默靶向mRNA，某些RNA病毒已经进化出 重新利用细胞miRNAs来稳定病毒基因组并刺激病毒基因的机制， 生产miRNA复合物被重定向以实现这些非经典的功能的机制是： 功能了解不多。我们正在通过调查互动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翻译以使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容易在动物细胞之间传递。这一发现的25年后，RNA的机制 动物细胞内的转运仍不清楚。我们正在通过确定 SID-1识别和转运RNA结构基础，一种广泛保守的整合膜 将双链核糖核酸运送到动物细胞中的蛋白质。预计这些综合研究将 提出克服上述主要知识障碍所需的见解， 了解人类健康，生育能力，病毒感染和基于RNA的疗法的进步。

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.