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复制的时机到转录的特异性等一系列重要过程。反过来，核和细胞质之间的物理通道，被称为核孔复合体，在正常的细胞功能中起着关键作用。了解通过毛孔的核质运输将实现细胞生物学的一个基本目标，并进一步有助于深入了解正常运输在病毒感染和细胞转化等条件下是如何出错的。目前，由于缺乏有关运输机制的信息，大分子核孔复合体的完整图景受到阻碍。因此，这项赠款提案侧重于了解毛孔蛋白如何引导和协调货物/受体复合体通过毛孔的运输的长期目标。提出的战略集中在探索一种特定的核孔素Nup153的功能，该核孔素是毛孔输出机制的核心组成部分。提出三个目标：(1)通过结构-功能分析，剖析Nup153 S在出口中的作用。将绘制Nup153内在与出口货物或出口机械部件对接方面重要的特定领域的地图。为了实现这一目标，将进行抗体干扰、显性阴性测试和突变分析。(2)将确定RNA结合在Nup153出口功能中的作用。通过测试Nup153片段的均聚物结合活性来描述与RNA结合的中介部位。将进行旨在区分与核糖核酸出口货物或核糖核酸孔组分相互作用的生化测试，以确定Nup153的生理核糖核酸靶标(S)。然后将评估扰乱这种互动的功能后果。(3)识别与重要的Nup153结构域相互作用的蛋白质。通过遗传和生化互作分析，Nup153将被用来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.