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 one of the largest macromolecular assemblies in the cell perforating the double membrane of the nuclear envelope. It is not known how NPCs form and insert into the nuclear membrane, and overall, the structure of the NPC remains poorly understood. NPCs function in the bidirectional transport of a very large set of diverse cargos. This includes the nuclear export of messenger RNAs, which is essential for the expression of every eukaryotic gene. Despite this fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC is ill defined. In addition to its role in nucleocytoplasmic transport, the NPC also functions in the regulation of multiple nuclear processes including the regulation of gene expression and the three-dimensional organization of the genome. The objectives of this research proposal are to elucidate the structure and assembly mechanism of the NPC and to characterize the roles of the NPC in messenger RNA export and in the organization of chromatin.
Our Specific Aims are: (1) To understand how mRNAs are directionally transported across the NPC. (2) To characterize the structure and biogenesis of NPCs. (3) To elucidate the function of the NPC in genome organization. We employ a combination of innovative biochemical, genetic and state-of-the-art single molecule imaging approaches in the single cellular eukaryote Saccharomyces cerevisiae to address these three specific aims. Yeast provides an outstanding model system to characterize the components and function of the NPC, to study NPC transport events in living cells, and to develop novel reconstitution assays. The NPC is a highly conserved structure, and mechanistic insights obtained from our studies will be directly relevant to all eukaryotes, including humans. Because NPC components are mutated in many diseases (e.g. cancer, heart diseases, or developmental disorders) and NPC function is disrupted by many viruses during infection to promote viral replication, our studies are also directly relevant for our understanding of human disease.
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.
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