Functional Analysis of Proteins Involved in Translational Regulation

Abstract

Cytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo.