FIGURE 5.

GLD2 is sufficient of promote Musashi-mediated polyadenylation and oocyte maturation. A, oocytes were injected with nuclease-free water (control) or mRNA mixtures containing GST-Musashi-1, HA-GLD2a, or GST-Musashi-1 and HA-GLD2a. The oocytes were allowed to express the injected RNA and were then stimulated to mature with progesterone. The maturation rate relative to control in five independent experiments is shown graphically. *, indicates p < 0.05; **, indicates p < 0.01. ns, not significant. B, representative Western blot showing protein expression from an experiment in A. C, representative poly(A) assay comparing polyadenylation of two endogenous mRNAs from Musashi/GLD2 co-injected oocytes relative to water-injected (control) oocytes. The Mos mRNA is under Musashi control and therefore polyadenylated early (prior to GVBD), whereas the cyclin B1 mRNA is under cytoplasmic polyadenylation element-binding protein control and polyadenylated late (at or after GVBD). The earliest signs of polyadenylation (*) appear at 3 h for all 3 conditions. − indicates pre-GVBD; + indicates post-GVBD samples. D, poly(A) length assay comparing polyadenylation of Mos mRNA from Musashi, GLD2, or water-injected (control) oocytes. The earliest signs of polyadenylation appear at 3 h for all three conditions. This indicates that the acceleration in onset of polyadenylation seen in C requires overexpression of both Musashi-1 and GLD2 together. − indicates pre-GVBD; + indicates post-GVBD. E, graphic representation of the poly(A) assay (C) for the Mos mRNA at the 3rd h. The dashed line is immature (Imm) oocytes (0 h), and the solid line is at the 3-h time point. Gray lines represent water (control) injection, and the black lines are Musashi/GLD2 co-injected oocytes. At the 3rd h, Musashi/GLD2 co-injection has promoted a polyadenylation of ∼30 adenylate residues, whereas control injected oocytes have only extended their poly(A) tails by ∼10 adenylates.