Poly(A) Removal and Translational Silencing of Maternal mRNAs

University Of Virginia Main Campus, Charlottesville VA

Investigators

Abstract

Oocytes synthesize and accumulate a large pool of maternal mRNAs. Many of these transcripts are actively utilized for protein synthesis throughout oogenesis, but are subsequently silenced when the oocyte completes meiosis and matures into an unfertilized egg, or when the egg is fertilized. As the oocyte has limited translational capacity, this silencing is necessary in order to permit the activation of other mRNAs encoding proteins essential for the completion of meiosis and early embryogenesis. The developmentally regulated removal of 3' poly(A) tails, a process termed deadenylation, is an evolutionarily conserved mechanism by which maternal mRNA are silenced in diverse organisms. This project addresses the molecular basis for the deadenylation-dependent translational inactivation of maternal mRNAs during the meiotic maturation of Xenopus oocytes. Deadenylation in mature oocytes is catalyzed by the poly(A)-specific ribonuclease, PARN. PARN activity is stimulated by the presence of the m7GpppG cap structure present at the 5' end of mRNAs, and cap recognition is an intrinsic property of this nuclease. By interacting directly with the 5' cap, PARN may also function in repressing translation initiation independently of its poly(A)-degrading activity. Competing interactions among PARN, the cap-binding translation initiation factor, eIF4E, and the poly(A) binding protein, PABP, provide a mechanism which integrates translational efficiency with poly(A) stability. This research utilizes complementary molecular biological and biochemical in vivo and in vitro approaches to address the following aspects of PARN-dependent deadenylation and translational silencing. First, to delineate functional domains required for PARN's poly(A) and cap-binding activities, and to address these aspects of PARN function in deadenylation. Second, this project will analyze functional and physical interactions between PARN and cap-dependent translation initiation factors. PARN deadenylation activity is intimately linked with translation efficiency as the 5' cap and 3' poly(A) tail are major determinants of both processes. These reactions are necessarily antagonistic as the relevant mRNA binding proteins, eIF4E and PABP, synergistically promote efficient translation initiation and inhibit PARN. In concert, these two objectives will define the mechanism of a novel poly(A)-specific ribonuclease and provide information on how translation initiation and poly(A) stability are coupled. Understanding the molecular basis of these evolutionarily conserved processes will provide insight into how maternal mRNAs are regulated to direct stage-specific programs of protein synthesis during early vertebrate development. As this translational regulation underlies many crucial events including the completion of meiosis, activation of the mitotic cell cycle, formation of body axes and the specification of cell fates, its elucidation is of fundamental importance and direct relevance to numerous problems in developmental biology and post-transcriptional gene regulation.