This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Communication between the nucleus and the 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 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 mRNA transport through the NPCs, we propose the following.
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 mRNA transport factors. To define the requirements for FG repeat numbers, FG types and critical FG binding sites in the NPC substructural locations, the mutants will be assayed for mRNA export defects. A biochemical approach will be used in aim two to detect changes in protein-protein interactions during the 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 the soluble inositol hexakisphosphate (IP6). 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 mRNA 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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