Gene expression in early animal development is controlled in part by the regulated translation of maternal mRNAs. The cytoplasmic polyadenylation element binding protein (CPEB) is one factorthat governs the translation of many of these mRNAs;it interacts with the 3' untranslated region (UTR) cytoplasmic polyadenylation element (CPE) to facilitate poly(A) shortening and translational repression as well as subsequent poly(A) elongation and translational activation. In mice with a disrupted CPEBgene, oocyte development is inhibited at the pachytene stage because of a failure to form synaptonemal complexes, two components of which are regulated at the translational level by CPEB. To investigate the importance of CPEB after pachytene, we will generate mice harboring a transgene composedof the zona pellucida 3 (ZP3) promoter followed by shRNA directed against CPEB mRNA. The destruction of CPEB mRNAwill take place after pachytene, thereby allowing an analysis of CPEB activity during the late stages of prophase I and during meiotic maturation. CPEB promotes polyadenylation through interactions with a number of other proteins, the most important of which are Gld2, an unusual poly(A) polymerase, and PARN,a deadenylase. Gld2 and PARN are both active and associatedwith the ribonucleoprotein (RNP) complex, however, PARNactivity is more robust and thus poly(A)tails are short. During oocyte maturation, CPEB phosphorylation results in the expulsion of PARNfromthe complex, resulting in Gld2-ctalyzed default polyadenylation. Several experiments will bedirected to understanding PARN activity in mouse oocytes, the most important of which will be the injection of catalytically inactive PARN,which may result in default Gld2 polyadenylation. If this occurs, PARNwill be knocked out in oocytes and the effects onoocyte maturation will be assessed. CPEB regulated translation involves an interaction with Maskin, which mediates the association of elF4G with elF4E, the cap-binding protein. Another protein with Maskin-like activities is Neroguidin (Ngd), which may be particularly important in mammals. The importance of Ngd-mediated translation in mouse oocyteswill be determined. Finally, mice havethree additional CPEB-like genes, one of which, CPEB3, is particularly prominently expressed in oocytes. Recently, ES cells with a disrupted CPEB3 gene have been injected into blastocysts and have been propagated through the gem line. The importance of CPEB3 for oocytedevelopment will be assessed. The proposed experiments will examine the molecular basis of oocyte development, and thus are relevant to human health especially human reproduction.
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