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.
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