The long-term objectives of this research program are to (1) define the mechanisms by which mRNA molecules are transported from the nucleus to the cytoplasm and (2) determine under what circumstances and by what mechanisms nucleocytoplasmic transport is regulated.
Three specific aims are proposed: 1. To determine the function in mRNA export performed by the DEAD-box protein Dbp5. Dbp5 shuttles and associates with nuclear pore complexes (NPCs). Full enzymatic activity of Dbp5 requires additional proteins but their identity is not known. Both biochemical and genetic approaches are proposed in order to identify proteins which interact with Dbp5 and to determine how they affect Dbp5 activity. Other studies are aimed at determining the mechanisms of Dbp5's nuclear import and export, including whether Dbp5 is exported with mRNA. Approaches include analysis of Dbp5's interactions with transport receptors and its localization in strains carrying mutations affecting receptors for nuclear import, export, or both. An essential task during mRNA export is the removal of proteins from the exported mRNA/protein complex. By remodeling the complex, packaging and export factors are released and recycled and new proteins associate with the mRNP, allowing it to be translated. An assay will be developed to determine whether Dbp5 can remove proteins from mRNA. 2. To determine the mechanisms responsible for regulation of mRNA export following heat or ethanol shock. Recent studies in this laboratory indicate that the promoter is responsible for determining whether or not an mRNA will be exported after stress. Studies are proposed to examine the hypothesis that differential export of mRNA following stress reflects regulated association of mRNA processing factors with RNA polymerase II. A genetic screen is proposed to determine the mechanism(s) responsible for nuclear retention of poly(A)+ RNA after stress. 3. To determine the roles in nuclear transport performed by Gfd1 and Gfd2. In a screen for high-copy suppressors of the ts dbp5-2 allele, GFD1 and GFD were identified. Neither are essential. Gfd1 shuttles and, like Dbp5, interacts with the Nupl59 subcomplex of NPCs, which appears to play a central role in mRNA export. Cells cannot survive if they carry the dbp5-2 allele and a disruption of GFD2. Studies are proposed to determine the functions of Gfdl and Gfd2, and to determine how their subcellular distribution is affected by mutation of several mRNA export factors.
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