The major objective of this proposal is to understand the molecular mechanisms that control gene expression at the posttranscriptional level in early vertebrate development. Prophase I-arrested Xenopus oocytes contain dormant (masked) mRNAs that have short poly(A) tails; when the oocytes re-enter the meiotic divisions during the process of maturation, the poly(A) tails are elongated and translation ensues. Cytoplasmic polyadenylation requires two cis elements in the mRNA 3' untranslated region: the cytoplasmic polyadenylation element (CPE), and the polyadenylation hexanucleotide AAUAAA. The CPE is bound by CPEB, which when phosphorylated during maturation, attracts cleavage and polyadenylation specificity factor (CPSF) to the AAUAAA; this factor in turn probably recruits poly(A) polymerase. Polyadenylation stimulates translation by causing the dissociation of maskin, a protein that is anchored to specific mRNAs via CPEB, from the cap binding factor elF4E. The release of maskin from elF4E allows elF4G to bind elF4E, which is necessary for the proper positioning of the 40S ribosomal subunit on the 5' end of the mRNA. Maskin undergoes a number of phosphorylation events during maturation, and the influence these modifications may have on translation will be examined. Polyadenylation-induced translation also occurs during the embryonic cell cycle, and the enzymes that are responsible for the oscillation of CPEB phosphorylation-dephosphorylation during this time will be analyzed. Cytoplasmic polyadenylation takes place during the somatic cell cycle as well; the necessity of polyadenylation for cell division and the factors that are responsible will be investigated. CPEB is the founding member of a family of RNA binding proteins that have a preference for the CPE; the functions of other CPEB-like proteins will be investigated. Maskin does not appear to be the only protein in Xenopus that interacts with elF4E; the function of a new maskin-like molecule will be investigated. Finally, a screen to identify the full array of mRNAs that undergo cytoplasmic polyadenylation will be initiated. Translational control by cytoplasmic polyadenylation is widely used in the animal kingdom, not only in early development, but in neurons and possibly somatic cells as well. Therefore, mechanistic studies of polyadenylation-induced translation are likely to have important implications for human health.
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