MOLECULAR MECHANISMS OF NUCLEAR EXPORT

细胞核内特殊的微环境对基因组调控至关重要，影响着一系列重要的过程，从DNA复制的时间到转录的特异性。反过来，细胞核和细胞质之间的物理通道，称为核孔复合物，在正常细胞功能中起着关键作用。了解核细胞质通过孔的运输将实现细胞生物学的一个基本目标，并进一步深入了解在病毒感染和细胞转化等条件下正常运输是如何出错的。目前，由于缺乏运输机制方面的信息，阻碍了对大分子核孔复合物的综合研究。因此，这项拨款提案的重点是了解孔蛋白如何指导和协调货物/受体复合物通过孔的运输的长期目标。提出的策略集中于探测一种特定核孔蛋白Nup153的功能，已知它是孔输出机制的核心组成部分。设想三个目标：(1)通过结构-功能分析剖析Nup153在出口中的作用。将映射Nup153中在与出口货物或出口机械组件接口方面很重要的特定域。抗体干扰，显性阴性试验和突变分析将进行，以实现这一目标。(2) RNA关联在Nup153输出功能中的作用将被确定。介导与RNA结合的位点将通过测试Nup153片段的同源聚合物结合活性来确定。设计用于区分与RNA出口货物或RNA孔组分相互作用的生化试验将进行，以确定Nup153的生理RNA靶标。然后将评估破坏这种相互作用的功能后果。(3)将鉴定出与功能重要的Nup153结构域相互作用的蛋白。通过基因和生化相互作用分析，Nup153将用于构建出口机械部件之间关键连接的更大图景。

The specialized microenvironment within the nucleus is critical to genomic regulation, impacting a range of important processes from the timing of DNA replication to the specificity of transcription. In turn, the physical gateway between the nucleus and cytoplasm, termed the nuclear pore complex, plays a key role in normal cell function. Achieving an understanding nucleocytoplasmic transport through the pore will fulfil a fundamental goal in cell biology and, further, will lend insight into how normal transport goes awry under conditions such as viral infection and cellular transformation. An integrated picture of the macromolecular nuclear pore complex is currently impeded by a lack of information regarding the mechanics of transport. This grant proposal is therefore focused on the long-term objective of understanding how pore proteins guide and coordinate the transit of cargo/receptor complexes through the pore. The strategy proposed centers on probing the function of a particular nucleoporin, Nup153, known to be a central component of the pore export machinery. Three goals are envisioned: (1) Nup153's role in export will be dissected through a structure-function analysis. The specific domains within Nup153 that are important in interfacing with either export cargo or components of the export machinery will be mapped. Antibody interference, dominant negative tests, and mutational analysis will be undertaken to achieve this goal. (2) The role of RNA association in the export function of Nup153 will be determined. The site that mediates association with RNA will be delineated by testing Nup153 fragments for homoribopolymer binding activity. Biochemical tests designed to distinguish between interactions with RNA export cargo or an RNA pore component will be performed to determine the physiological RNA target(s) of Nup153. Functional consequences of disrupting this interaction will then be assessed. (3) Proteins that interact with functionally important Nup153 domains will be identified. Through genetic and biochemical interaction analyses, Nup153 will be used to construct a bigger picture of key connections made between export machinery components.