Structure and function of the nuclear pore complex

描述（由申请人提供）：核孔复合物的结构和功能对于所有真核生物的细胞内组织至关重要的是大分子在细胞核和细胞质之间的精确转运，这是通过核孔复合物（NPC）的功能实现的。NPC是一个巨大的运输通道，贯穿核膜的双层膜。然而，NPC如何形成以及它们如何插入核膜尚不清楚。NPC在许多不同的运输事件中起着重要作用，包括信使RNA从细胞核到细胞质的输出。但是，尽管它的基本意义，信使RNA通过NPC定向易位的途径仍然不清楚。本研究的目的是阐明NPC在核膜中形成和组装的机制，并描述NPC在信使RNA输出中的作用。本提案中描述的所有实验方法都利用了可用于单细胞真核生物酿酒酵母（Saccharomyces cerevisiae）的蛋白质组学、基因组学和细胞生物学工具。酵母提供了一个很好的模型系统来表征NPC的组分和功能，研究活细胞中复杂的NPC组装过程，并开发新的重建测定。我们的目标是：（1）研究鼻咽癌组织中Gle1和Dbp5基因在mRNA输出中的作用，探讨mRNA在鼻咽癌组织中的单向转运。(2)研究跨膜核孔蛋白Ndc1在鼻咽癌细胞组装和结构中的作用。(3)使用基于荧光的测定来检查活细胞中的NPC生物合成，并使用确定的组分在体外重建NPC组装步骤。我们采用创新的生物化学，遗传学和细胞生物学方法相结合，以解决这三个具体目标，在S。啤酒。由于NPC是一个高度保守的结构，从这些研究中获得的机制见解将直接与所有真核生物，包括人类。公共卫生相关性：在所有的真核生物中，通过核孔复合物的大分子运输的调节提供了一个重要的机制，信号转导途径和发育刺激控制差异基因表达。此外，许多病毒靶向细胞核转运机制的组分以促进病毒繁殖，并且几种致癌易位涉及核孔复合物的组分以促进癌症发展。因此，更好地了解介导核质转运的分子机制对于理解基本细胞过程和开发新的抗病毒和抗癌疗法都是至关重要的。

DESCRIPTION (provided by applicant): STRUCTURE AND FUNCTION OF THE NUCLEAR PORE COMPLEX 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 an enormous transport channel that perforates the double membrane of the nuclear envelope. However, how NPCs form and how they are inserted into the nuclear membrane is not known. NPCs play an essential role in many diverse transport events including the export of messenger RNAs from the nucleus to the cytoplasm. But despite its fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC remains poorly defined. The goals of this research proposal are to elucidate the mechanism by which NPCs form and assemble in the nuclear envelope and to characterize the role of the NPC in messenger RNA export. All experimental approaches described in this proposal take advantage of the proteomic, genomic and cell biological tools available for use within the single cellular eukaryote Saccharomyces cerevisiae. Yeast provides an excellent model system to characterize the components and function of the NPC, to study the complex NPC assembly process in living cells, and to develop novel reconstitution assays. Our aims are: (1) To characterize the role of the NPC components Gle1 and Dbp5 in mRNA export and to investigate how mRNAs are unidirectionally transported across the NPC. (2) To study the role of the transmembrane nuclear pore protein Ndc1 in NPC assembly and structure. (3) To use fluorescence based assays to examine NPC biosynthesis in living cells and to reconstitute NPC assembly steps in vitro using defined components. We employ a combination of innovative biochemical, genetic and cell biological approaches to address these three specific aims in S. cerevisiae. Because the NPC is a highly conserved structure, the mechanistic insights obtained from these studies will be directly relevant to all eukaryotes, including humans. PUBLIC HEALTH RELEVANCE: In all eukaryotes, regulation of macromolecular transport through the nuclear pore complex provides an essential mechanism by which signal transduction pathways and developmental stimuli control differential gene expression. In addition, many viruses target components of the cellular nuclear transport machinery to facilitate viral propagation and several oncogenic translocations involve components of the nuclear pore complex to promote cancer development. Therefore, a better understanding of the molecular machinery that mediates nucleocytoplasmic transport is essential both for understanding fundamental cellular processes and the development of novel anti-viral and anti-cancer therapies.