Figure 3

The 3'UTR of zebrafish cyclinB1 mRNA is sufficient to activate translation during zebrafish oocyte maturation and this activation requires the CPE and AAUAAA sequences. (A) Schematic of various luciferase reporter mRNAs with different 3'UTRs (ZF-zebrafish). (B) The zebrafish cyclin B1 3'UTR directs translational activation during oocyte maturation and activation requires the putative CPE elements and AAUAAA element. Luciferase reporter mRNAs with various zebrafish cyclinB1 3'UTRs were injected into oocytes. Some of the injected oocytes were matured. Luciferase activity was measured in extracts prepared from injected oocytes and injected matured oocytes. The ratio of luciferase in matured versus non-matured oocytes was plotted for comparison. The mat/non-mature ratios shown are represented as mean +/- SEM of at least three independent experiments. Statistical analysis (P values) was performed by one-way ANOVA. * P < 0.05. Each reporter containing zebrafish cyclin B1 mutant 3'UTR, bactin2 3'UTR or no 3'UTR was compared to the reporter with zebrafish cyclin B1 WT 3'UTR. (C) Luciferase reporters were equally stable in mature vs. non-mature oocytes. Total RNA was extracted from mature and non-matured oocytes injected with each zebrafish cyclin B1 3'UTR reporter mRNA. Equal amounts of total RNA from each sample were utilized in quantitative RT-PCR using luciferase-specific primers. Product formation was monitored with increasing cycles of amplification and analyzed by agarose gel electrophoresis. (D) The presence of a poly (A) tail is sufficient to stimulate mRNA translation in zebrafish oocytes. Luciferase reporter mRNAs (Luc vs. LucA50) were injected into 20–30 fully-grown zebrafish oocytes and assayed for luciferase activity after 6 hours. This experiment was repeated twice and shown here are the absolute values of luciferase activity from one representative experiment.