Translational control is an important regulatory mechanism in early development. Synthesis of required proteins is kept in check until they are needed, avoiding inappropriate activation of signaling pathways and erroneous development. Translational regulation of essential mRNAs during development is mediated by maskin-eIF4E interaction, but otherwise is not well understood. The long-term goals of this study are to determine how this translational masking and unmasking are regulated, focusing on the roles of phosphorylation and cytoplasmic polyadenylation. Mapping of maskin phosphorylations both during translational masking and those that occur during translational unmasking will allow examination of how these modifications affect eIF4E binding. Mutations of these sites will also facilitate study of maskin translational repression in vivo using a MS2 coat-protein maskin hybrid. This will tether maskin to the mRNA and allow for translation to be monitored in the presence or absence of a poly(A) tail. To further examine the hypothesis that cytoplasmic polyadenylation is essential unmasking, maturation will be induced by the infection of mos or MPF while blocking polyadenylation with cordycepin. Maskin binding to eIF4E will be monitored under these conditions. Additionally, in vitro reconstitution of cytoplasmic polyadenylation will be coupled to in vitro translational masking in the presence of wild type and phosphorylation mutants of maskin allowing biochemical analysis of masking and unmasking and the role of polyadenylation.
|Barnard, Daron C; Cao, Quiping; Richter, Joel D (2005) Differential phosphorylation controls Maskin association with eukaryotic translation initiation factor 4E and localization on the mitotic apparatus. Mol Cell Biol 25:7605-15|
|Barnard, Daron C; Ryan, Kevin; Manley, James L et al. (2004) Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. Cell 119:641-51|