? Communication between the nucleus and cytoplasm is mediated by large proteinaceous structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). The long-term goal of this project is to elucidate the molecular sequence of events required for translocation through the NPC. We hypothesize that the framework for selective, facilitated NPC translocation is based on physical interactions between shuttling transport factors and a family of NPC proteins that contain regions with multiple FG-type repeats (F, phenylalanine; G, glycine). We also propose that key events for regulation of mRNA export directionality are controlled at the NPC by the action of multiple factors. To analyze the mechanism and regulation of protein and mRNA transport through NPCs, we propose three aims.
In aims one and two, we will build on our recent results documenting the first in vivo tests of NPC translocation models. We propose to use S. cerevisiae mutants with minimal repertoires of the FG binding sites for protein and mRNA transport factors. To define the requirements for FG repeat numbers, FG types and critical FG binding sites in NPC substructural locations, the mutants will be assayed for protein import and mRNA export defects. NPC-transport factor association will also be analyzed using microscopy strategies. A biochemical approach will be used in aim two to detect changes in protein-protein interactions during extrusion of the mRNA-protein complex through the NPC.
In aim three, we will investigate the mechanism for activation of the DEAD-box helicase Dbp5 by the essential mRNA export factor Gle1 and production of soluble inositol hexakisphosphate (IPS). We will identify the IP6 binding site in Gle1/Dbp5, and test for roles in RNA unwinding and remodeling of RNA- protein complexes. Studies in yeast and human tissue culture cells will also be conducted to test for regulation of Gle1 and IP6 in other aspects of mRNA processing. This proposal represents an area of basic science research that has the potential to provide novel insights into multiple disease processes. Transport factors and NPC proteins are targets for viral inhibition of cell function and mediators of viral RNA export. Inositol signaling defects are associated with disease states including cancer cell growth, inflammation, neurotransmission, and organ development. Knowledge of the NPC translocation mechanism will be key for designing therapeutic strategies to selectively target these pathways. ? ? ?
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