The regulation of maternal mRNA expression during oocyte maturation in Xenopus laevis will be examined. In particular, it has been demonstrated that poly(A) elongation of several mRNAs is critically important for their translation during maturation. The cis (nucleotide sequence) and trans (binding proteins) elements that control this polyadenylation have been identified. Moreover, there is ample evidence that the phosphorylation of an RNA binding protein by the kinase, p34(cdc2), regulates polyadenylation. The objectives of this proposal are to study the trans factors in detail and to assess their regulation by p34(cdc2) kinase. The trans factors will be isolated biochemically and will be used to obtain limited amino acid sequence information for the synthesis of oligonucleotide probes based and the screening of a cDNA library. The cDNA clones, in turn, will be used for overexpression of protein in baculovirus. The baculovirus-expressed proteins will be tested for activity in polyadenylation-competent extracts, analyzed for phosphorylation by baculovirus-expressed p34(cdc2) kinase, and used to determine how its interaction with RNA and possibly other proteins induces polyadenylation. The significance of these studies is underscored by the fact that they will bring together the fields of translational control, RNA processing, and cell cycle control. As such, the proposal has relevance to the fields of growth control (cancer) and early development.
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