Structure and function of the nuclear pore complex

描述(申请人提供)：对所有真核生物的细胞内组织来说，最重要的是大分子在细胞核和细胞质之间的准确运输，这是通过核孔复合体(NPC)的功能实现的。鼻咽癌是穿透核膜双层膜的细胞中最大的大分子集合体之一。目前尚不清楚NPC是如何形成和插入核膜的，总的来说，NPC的结构仍然知之甚少。NPC在大量不同货物的双向运输中发挥作用。这包括信使RNA的核出口，这对每一个真核基因的表达都是必不可少的。尽管有这一基本意义，信使RNA通过鼻咽癌定向转移的途径尚不明确。鼻咽癌除了在核质转运中发挥作用外，还参与多种核过程的调控，包括基因表达调控和基因组三维结构的调控。这项研究的目的是阐明NPC的结构和组装机制，并表征NPC在信使RNA输出和染色质组织中的作用。我们的具体目标是：(1)了解mRNAs是如何在NPC中定向运输的。(2)研究鼻咽癌细胞的结构和生物发生。(3)阐明鼻咽癌在基因组组织中的作用。我们在单细胞真核细胞酿酒酵母中采用了创新的生化、遗传和最先进的单分子成像方法来解决这三个特定目标。酵母菌为研究鼻咽癌的组成和功能、研究活细胞中的鼻咽癌转运事件以及开发新的重组分析方法提供了一个出色的模型系统。NPC是一个高度保守的结构，从我们的研究中获得的机械论见解将直接与包括人类在内的所有真核生物相关。由于NPC在许多疾病(如癌症、心脏病或发育障碍)中会发生突变，并且在感染过程中会被许多病毒干扰以促进病毒复制，因此我们的研究也与我们对人类疾病的理解直接相关。

DESCRIPTION (provided by applicant): Of central importance to the intracellular organization of all eukaryotes is the accurate transport of macromolecules between the nucleus and the cytoplasm, which is achieved through the function of the nuclear pore complex (NPC). The NPC is one of the largest macromolecular assemblies in the cell perforating the double membrane of the nuclear envelope. It is not known how NPCs form and insert into the nuclear membrane, and overall, the structure of the NPC remains poorly understood. NPCs function in the bidirectional transport of a very large set of diverse cargos. This includes the nuclear export of messenger RNAs, which is essential for the expression of every eukaryotic gene. Despite this fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC is ill defined. In addition to its role in nucleocytoplasmic transport, the NPC also functions in the regulation of multiple nuclear processes including the regulation of gene expression and the three-dimensional organization of the genome. The objectives of this research proposal are to elucidate the structure and assembly mechanism of the NPC and to characterize the roles of the NPC in messenger RNA export and in the organization of chromatin. Our Specific Aims are: (1) To understand how mRNAs are directionally transported across the NPC. (2) To characterize the structure and biogenesis of NPCs. (3) To elucidate the function of the NPC in genome organization. We employ a combination of innovative biochemical, genetic and state-of-the-art single molecule imaging approaches in the single cellular eukaryote Saccharomyces cerevisiae to address these three specific aims. Yeast provides an outstanding model system to characterize the components and function of the NPC, to study NPC transport events in living cells, and to develop novel reconstitution assays. The NPC is a highly conserved structure, and mechanistic insights obtained from our studies will be directly relevant to all eukaryotes, including humans. Because NPC components are mutated in many diseases (e.g. cancer, heart diseases, or developmental disorders) and NPC function is disrupted by many viruses during infection to promote viral replication, our studies are also directly relevant for our understanding of human disease.