Orb and Wispy cooperate in the cytoplasmic polyadenylation of grk mRNA during oogenesis

Abstract

During Drosophila oogenesis, the TGF-alpha like protein Gurken (Grk) is required for establishing both of the major axes of the egg and future embryo. Consequently, Grk protein distribution within the ovary is tightly regulated, both temporally and spatially. It is well established that Grk protein distribution in the oocyte is regulated by a combination of the localization of the grk transcript and regulation of grk mRNA translation. While it is clear that grk translation is both repressed, when the mRNA is not properly localized and activated, once the transcript has reached its destination in the dorsal anterior corner of the oocyte, the molecular mechanism(s) of grk mRNA translational control have not yet been fully elucidated. Modulating polyAtail length is one mechanism for regulating translation of other mRNAs and is accomplished through cytoplasmic deadenylation and polyadenylation of transcripts. We have therefore investigated whether translational control of grk RNA is achieved through alterations in polyA tail length. Genetic evidence has shown that the cytoplasmic polyadenylation element binding (CPEB) protein Orb is required for appropriate Grk protein accumulation, suggesting that cytoplasmic polyadenylation of grk mRNA may have a role in its translational activation. Using a PCR-based polyadenylation assay, we have analyzed grk transcript polyadenylation status directly. We have found that grk polyA tail length is indeed shorter in transcripts isolated from orb mutant females. Additionally, females carrying mutations in wispy (wisp), a GLD-2 polyA polymerase lay eggs with slight patterning defects that are consistent with suboptimal levels of Grk protein at the D/A corner of the oocyte. grk mRNA is also underadenylated in ovaries from wisp mutant mothers. Finally, mutations in wisp enhance the ventralizing effects of a dominant negative orb allele, suggesting that Orb and Wisp function together in the cytoplasmic polyadenylation of grk mRNA once it has reached the dorsal anterior corner of the oocyte. Our current efforts are aimed at assessing whether the translational repression of unlocalized grk mRNA is also achieved through regulated deadenylation-adenylation. Initial experiments indicate that translationally repressed grk transcripts, for example in spnF oocytes or associated with the RNAbinding protein Squid (Sqd), are underadenylated. These findings suggest that regulation of Grk protein accumulation is, in part, achieved through polyA tail length alterations.