Figure 4

Directing polyadenylation and activating translation during oocyte maturation are evolutionarily conserved functions of cyclin B1 3'UTRs. (A) The zebrafish cyclinB1 3'UTR is sufficient to direct polyadenylation during Xenopus oocyte maturation, and the CPEs are required. Each P³²-labeled 3'UTR RNA was injected into 30–50 Xenopus oocytes and some oocytes were matured. RNA isolated from injected non-mature oocytes or injected matured oocytes were analyzed by 4% denaturing PAGE. IN: Uninjected RNA. N: RNA from non-mature oocytes. M: RNA from mature oocytes. Half of the RNA from WT 3'UTR injected mature oocytes was treated with oligo dT/RNaseH prior to analysis (lane 4). The size reduction after oligo dT/RNaseH treatment (lane 4 versus lane 3) indicated that the WT 3'UTR RNA was polyadenylated. (B) The Xenopus cyclinB1 3'UTR is sufficient to direct polyadenylation during zebrafish oocyte maturation, and the CPEs are required. Each P³²-labeled 3'UTR RNA was injected into 50–100 zebrafish oocytes and some oocytes were matured. RNA from injected non-matured oocytes or injected matured oocytes were analyzed by 4% denaturing PAGE. IN: Uninjected RNA. N: RNA from non-mature oocytes. M: RNA from mature oocytes. Half of the RNA extracted from WT 3'UTR injected matured oocytes was treated with oligo dT/RNaseH prior to analysis (lane 4). The size reduction after oligo dT/RNaseH treatment indicated that the WT 3'UTR RNA was polyadenylated (compare lanes 3 and 4). (C) The zebrafish cyclinB1 3'UTR is sufficient to activate translation during Xenopus oocyte maturation and the AAUAAA and CPE sequences are required for this activation. Luciferase reporter RNAs with various 3'UTRs were injected into 30–40 Xenopus oocytes and half were matured with progesterone. The ratios of luciferase activities from mature vs. non-mature oocytes were calculated and graphed as mean +/- SEM from at least three independent experiments. Statistical analysis was performed by one-way ANOVA. * P < 0.05, ** P < 0.01. Each Xenopus mutant 3'UTR was compared to Xenopus WT 3'UTR, each zebrafish mutant cyclin B1 3'UTR or WT bactin2 3'UTR was compared to zebrafish WT cyclin B1 3'UTR. The mature/non-mature ratio of Xenopus cyclin B1 3'UTR is significantly different from that of zebrafish cyclin B1 3'UTR (D) The Xenopus Cyclin B1 3'UTR is sufficient to activate translation during zebrafish oocyte maturation and the AAUAAA and CPE sequences are required for this activation. Luciferase reporter RNAs with various 3'UTRs were injected into 50–100 zebrafish oocytes and half were matured with hormone. The ratio of luciferase activities from mature versus non-mature oocytes were calculated and graphed as the mean values +/- SEM from at least three independent experiments. Statistical analysis was performed by one-way ANOVA. * P < 0.05. Each mutant Xenopus reporter was compared to WT Xenopus reporter. The mature/non-mature ratio of zebrafish cyclin B1 3'UTR is significantly different from that of Xenopus cyclin B1 3'UTR.