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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033998-17
Application #
6400758
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Chin, Jean
Project Start
1984-08-01
Project End
2005-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
17
Fiscal Year
2001
Total Cost
$430,341
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Estruch, Francisco; Hodge, Christine; Gomez-Navarro, Natalia et al. (2012) Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors. BMC Genet 13:80
Hodge, Christine A; Tran, Elizabeth J; Noble, Kristen N et al. (2011) The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1. Genes Dev 25:1052-64
Noble, Kristen N; Tran, Elizabeth J; Alcázar-Román, Abel R et al. (2011) The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1. Genes Dev 25:1065-77
Folkmann, Andrew W; Noble, Kristen N; Cole, Charles N et al. (2011) Dbp5, Gle1-IP6 and Nup159: a working model for mRNP export. Nucleus 2:540-8
Hodge, Christine A; Choudhary, Vineet; Wolyniak, Michael J et al. (2010) Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum. J Cell Sci 123:141-51
Schneiter, Roger; Cole, Charles N (2010) Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins. Nucleus 1:387-92
Estruch, Francisco; Peiró-Chova, Lorena; Gómez-Navarro, Natalia et al. (2009) A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor. Mol Genet Genomics 281:125-34
Scarcelli, John J; Viggiano, Susan; Hodge, Christine A et al. (2008) Synthetic genetic array analysis in Saccharomyces cerevisiae provides evidence for an interaction between RAT8/DBP5 and genes encoding P-body components. Genetics 179:1945-55
Rollenhagen, Christiane; Hodge, Christine A; Cole, Charles N (2007) Following temperature stress, export of heat shock mRNA occurs efficiently in cells with mutations in genes normally important for mRNA export. Eukaryot Cell 6:505-13
Scarcelli, John J; Hodge, Christine A; Cole, Charles N (2007) The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes. J Cell Biol 178:799-812

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