MOLECULAR MECHANISMS OF NUCLEAR EXPORT

细胞核内的特殊微环境对基因组调控至关重要，影响着从DNA复制的时机到转录特异性的一系列重要过程。 反过来，细胞核和细胞质之间的物理通道，称为核孔复合体，在正常细胞功能中起着关键作用。实现理解核质运输通过孔将实现细胞生物学的一个基本目标，并进一步，将提供洞察正常的运输是如何出错的条件下，如病毒感染和细胞转化。一个完整的大分子核孔复合物的图片是目前阻碍了缺乏信息的机制，运输。因此，这项资助计划的重点是了解孔蛋白如何引导和协调货物/受体复合物通过孔的运输的长期目标。提出的策略集中在探测特定核孔蛋白Nup 153的功能上，Nup 153已知是孔出口机制的中心组成部分。本研究的目的有三：（1）通过结构-功能分析，探讨Nup 153在输出中的作用。Nup 153中与出口货物或出口机械组件接口的特定域将被映射。 将进行抗体干扰、显性阴性试验和突变分析以实现这一目标。(2)将确定RNA缔合在Nup 153的输出功能中的作用。将通过测试Nup 153片段的同核糖聚合物结合活性来描绘介导与RNA结合的位点。将进行旨在区分与RNA输出货物或RNA孔组分相互作用的生化试验，以确定Nup 153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)将鉴定与功能上重要的Nup 153结构域相互作用的蛋白质。通过遗传和生化相互作用分析，Nup 153将用于构建出口机械部件之间关键联系的更大图景。

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.