Regulated mRNA translation is used by metazoans to determine when and where the encoded proteins will be synthesized. mRNAs for cell cycle regulatory proteins and proto-oncogenes constitute one group whose temporal expression is under translational control. The expression of one particular proto-oncogene, c-mos, is regulated virtually completely at the translational level. In oocytes, c-mos mRNA is translationally dormant, but is activated when the cells re-enter the meiotic divisions. In mice, Mos is essential for metaphase II arrest; in transgenic animals lacking the c-mos gene, oocytes divide parthenogenetically and induce ovarian teratomas. The translation of c-mos mRNA is regulated by polyadenylation; when the poly(A) tail is short in oocytes, the message is dormant, when the tail is extended during meiotic maturation, the message is translated. Two cis elements in the 3' untranslated region of c-mos mRNA control polyadenylation; the hexanucleotide AAUAAA, and a U-rich specificity sequence called the cytoplasmic polyadenylation element (CPE). Initial experiments using Xenopus oocytes found that the CPE is bound by a protein CPEB, that regulates polyadenylation and resulting translation. Mouse oocytes also contain CPEB (mCPEB) where it binds the c-mos CPE. The major goal of this proposal center around the function of mCPEB in mouse oocytes. The role of CPEB in c-mos mRNA polyadenylation will be examined by injecting oocytes with antisense oligonucleotides to destroy mCPEB mRNA, as well as by injecting oocytes with mRNAs encoding dominant negative forms of mCPEB. The function of mCPEB phosphorylation will be assessed by mapping the sites of phosphorylation mutating them to alanine, and analyzing the effects on c-mos mRNA binding, polyadenylation, and translation. The mechanism by which polyadenylation induces translation will be examined, particularly with respect to cap-specific 2'-O-methylation. Finally, an mRNA that is deadenylated and stored in a dormant form in mouse oocytes undergoes prior CPE-mediated deadenylation. The possible role of mCPEB in the process will be assessed.
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