This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of the mRNA cap-binding protein eIF4E, which is involved in recruitment of mRNA to the ribosome. This process determines the rate of protein synthesis, the spectrum of mRNAs translated, and the rate of mRNA turnover. The project centers on two aspects of eIF4E: 1) the role of its phosphorylation and 2) proteins that bind to it. For the first, although the rate of protein synthesis and phosphorylation of eIF4E are highly correlated, the biochemical consequences of eIF4E phosphorylation remain elusive and controversial. The most definitive data will come from genetically tractable animal models like C. elegans. We have discovered and extensively characterized five family members of eIF4E in C. elegans, termed IFE-1, IFE-2, etc. We propose to determine the phosphorylation sites in each of the five IFEs by mass spectrometry. Then we will alter the site in one particular IFE to prevent phosphorylation and determine the result of expressing the modified form in knockout worms with regard to protein synthesis, spectrum of mRNAs translated, and overall phenotype of the organism. For the second, at least one of the IFEs is specifically bound to a protein, PGL-1, resident in P-granules, which are markers of the germline. In other systems, eIF4E-binding proteins are responsible for biological actions of eIF4E. We propose to identify proteins that co-purify with individual IFE family members by performing m7GTP-Sepharose affinity chromatography, comparing wild-type C. elegans with strains lacking each of the five IFEs.
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