The Developmentally Important RNA‐binding Protein, Zygote arrest (Zar), Regulates mRNA Translation

Zygote arrest (Zar) proteins, Zar1 and Zar2, are required for successful fertilization and embryogenesis. Zar proteins bind mRNA sequence specifically, via the Translation Control Sequence (TCS), a cis ‐element in the 3′UTR of mRNAs that regulates translation during early development. The TCS represses translation in the immature egg and activates translation and cytoplasmic polyadenylation in the maturing egg. Our objective is to show that Zar proteins are bona fide translation factors and are candidates for mediating translational regulation by the TCS. A dual‐luciferase reporter tethered assay was used to measure the effect on translation by Zar proteins. RNA ligation‐coupled RT‐PCR was used to evaluate Zar‐mediated cytoplasmic polyadenylation. GST affinity purification and mass spectrometry were used to identify proteins that interact with Zar and interactions were verified by co‐immunoprecipitation (Co‐IP). We show that when tethered to mRNA reporters, both Zar proteins repressed translation up to 50% and this repression was relieved during egg maturation, although no translational activation and no polyadenylation of the mRNA reporters were observed. Zar1 required a polyA tail to repress translation, whereas Zar2 did not. Zar1 and Zar2 interacted with overlapping but distinct sets of proteins and some interactions changed during egg maturation. Proteins recovered from co‐IPs included many known translation factors, such as: CPEB, Rap55, DDX6, and embryonic polyA‐binding protein. Together, these data suggest Zar proteins have a role as translation regulators and they may mediate repression by the TCS, but are not likely to mediate translation activation and cytoplasmic polyadenylation. They also suggest mechanistic differences between Zar1 and Zar2.