Figure 2

Polyadenylation of the zebrafish cyclin B1 mRNA 3'UTR depended upon U-rich CPE sequences. (A) Sequences of wild-type, AAUAAA mutant and U-rich mutant zebrafish cyclin B1 3'UTRs. The zebrafish cyclin B1 3'UTR is 199 nucleotides in length and the last 99 nucleotides are shown. U-rich putative CPE elements adjacent to AAUAAA are overlined and bolded while the AAUAAA sequence is underlined. Mutated sequences are white on black. Other U-rich sequences are present in the zebrafish cyclin B1 3'UTR, but their distance from AAUAAA makes it unlikely that they function as CPEs. (B) Sequences of wild-type, AAUAAA mutant and CPE mutant Xenopus cyclinB1 3'UTRs. Mutated sequences are white on black, CPE elements are overlined and bolded and AAUAAA elements are underlined. The numbers indicate positions of each nucleotide relative to the poly (A) addition site. (C) Zebrafish oocytes and matured oocytes. (D) Zebrafish cyclin B1 3'UTR is sufficient to direct polyadenylation during oocyte maturation, and the CPEs are required. P³²-labeled RNA consisting of the zebrafish cyclin B1 mRNA 3'UTR was injected into 50–100 zebrafish oocytes and some oocytes were matured. RNA from injected non-mature oocytes (N) or injected matured oocytes (M) were analyzed by 4% denaturing PAGE and autoradiography. IN: Uninjected RNA. N: RNA from non-mature oocytes. M: RNA from mature oocytes. As controls, half of the total RNAs extracted from WT 3'UTR RNA injected matured oocytes were subjected to oligo dT/RNaseH treatment prior to denaturing PAGE (lane 4). The size reduction after oligo dT/RNaseH treatment indicated that the 3'UTR RNA was polyadenylated.