Regulation of Eukaryotic Protein Synthesis Initiation

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DESCRIPTION (provided by applicant): The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of initiation factors that recruit mRNA to the ribosome, a process that determines rate of protein synthesis, spectrum of mRNAs translated, and rate of mRNA turnover. The research is centered on the mRNA cap-binding protein elF4E, one mechanism by which elF4E is regulated, the role of elF4Ein mRNA turnover, and elF3, the factor that links elF4E (via elF4G) to the 40S ribosomal subunit. Aim 1 is to elucidate the physiological roles of three of the five elF4E isoforms (termed IFEs) in C. elegans. We will use bioinformatics and biochemical approaches to discover the functions of IFE-1, -2, and -4 including genome wide analysis of mRNAs whose translation is dependent on each IFE. We will correlate IFE-specific translation with mRNA structural features and with IFE-specific binding proteins. Aim 2 is to examine the function of elF4E phosphorylation and its putative kinase, MNK-1, in C. elegans. We will attempt to resolve contradictory reports by determining the effect of IFE-2 phosphorylation on translation of IFE-2-dependent mRNAs and phenotypic traits, and we will characterize the putative C. elegans MNK-1 at the enzymological and whole animal levels. In Aim 3, we will investigate the role of elF4E in mRNA turnover in cultured mammalian cells by synthesizing novel cap analogs that are resistant to in vivo decapping and by altering the competition for cap binding between elF4E and Dcp1/Dcp2. In Aim 4, we will follow up on our observation that elF3e, one of the subunits of mammalian elF3, forms at least part of the binding site for elF4G. We will biochemically characterize the elF4G-elF3e interaction and role of elFSe in translation. We will also attempt to learn the mechanism by which MMTV insertion into the elFSe gene causes mammary tumors in mice. These studies have relevance to cancer because overexpression of elF4E causes malignant transformation of cells, human tumors contain greatly elevated levels of elF4E, and elF4E is the target of novel anti-cancer therapies. Also, MMTV integration into the elFSe gene is one of the best developed experimental cancer models. Furthermore, elF4E plays a critical role in both germ cell maturation and early embryonic development, which impacts on abnormalities resulting in birth defects. Finally, one elF4E isoform is required for normal spermatogenesis, which may shed light on infertility conditions.