9808196 WORMINGTON Oocytes synthesize and accumulate a large pool of maternal mRNAs. Some of these are actively translated throughout oogenesis while others remain quiescent until they are activated or "unmasked" at subsequent developmental stages. This translational control is the principal mechanism to regulate protein synthesis before the onset of zygotic transcription. Extensive analyses have established that the 3' untranslated region (3' UTR) is a critical determinant of maternal mRNA translation in organisms as diverse as nematodes, flies, clams, frogs and mice. In many cases the 3' UTRs of maternal mRNAs activate or repress translation by directing the stage-specific addition or removal of 3' poly(A) tails. This project addresses the molecular basis for the deadenylation-dependent translational inactivation of two distinct classes of maternal mRNAs during the meiotic maturation and subsequent fertilization of Xenopus oocytes. One class of mRNAs is polyadenylated and translated throughout oogenesis, but is deadenylated and translationally silenced at maturation. Poly(A) removal in mature oocytes does not require specific cis-sequences and is a default pathway of translational inactivation. Conversely, another class of mRNAs is not translated before progesterone-induced maturation. These mRNAs contain a specific 3' UTR sequence termed the cytoplasmic polyadenylation element (CPE) which is required for their poly(A)-elongation and translational activation. The presence of a CPE is sufficient to prevent default deadenylation in mature oocytes. However, a subset of CPE-mRNAs also contain a discrete 3' UTR sequence termed the embryonic deadenylation element (EDEN) which promotes their deadenylation at fertilization and thereby restricts their translation to unfertilized eggs. An intrinsically poly(A)-specific deadenylating nuclease (DAN) that catalyzes default poly(A) removal in mature Xenopus oocytes has been purified. A human cDNA encoding an immunocrossreactive and catalytically equivalen t poly(A) nuclease has been isolated using peptide sequences derived for the purified Xenopus DAN. This project will utilize immunological and nucleic acid reagents derived from the human DAN and a combination of molecular biological and biochemical approaches to address the following aspects of poly(A) removal and maternal mRNA translational inactivation in Xenopus oocytes and embryos: First, to investigate the developmental regulation of the Xenopus DAN. Antibodies and cDNAs corresponding to two DAN isoforms will be used to examine their expression during oogenesis and embryogenesis. These initial studies will be extended to establish the causal relationships between stage-specific changes in the synthesis, post-translational modification and nuclear-cytoplasmic distribution of DAN isoforms that correlate with alterations in deadenylation activity. Second, to determine if the Xenopus DAN that catalyzes default deadenylation in mature oocytes also participates in the sequence-specific deadenylation of EDEN-containing mRNAs in embryos. Complementary approaches will ascertain interactions between DAN and an EDEN-specific RNA binding protein. Third, to initiate the delineation of functional domains within Xenopus DAN required for its poly(A)-specific 3' exonucleolytic activity and developmental regulation. During egg maturation, the expression of many genes is controlled at the level of protein synthesis. The synthesis of a specific protein is inhibited by the removal of a tract of polyadenosine located at the 3' end of the mRNA, which can be regulated by any one of several pathways. This project will examine various aspects of this regulatory system, including a characterization of the deadenylating nuclease that removes the poly(A). Understanding the molecular basis of this regulatory process will provide insight into how egg mRNAs direct regulated protein synthesis during the early steps of vertebrate development. Their elucidation is of fundamental importance to numerous problems in de velopment biology and post-transcriptional gene regulation.
