UGCAUG repeats enhance exon 16 splicing and activate DUP internal exon splicing in a differentiation stage–specific manner in MELCs. (A) Schematic of the wild-type (WT) exon 16 minigene and mutations tested. The first repeat was mutated to restriction site PmlI, the second repeat to NruI, and the third repeat to BssSI. The double-mutant PN construct incorporated both the Pm and Nr mutations; the triple mutant PNB construct incorporated all 3 mutations. (B) Mutational analysis in undifferentiated and differentiated MELCs. Minigene stably transfected MELCs were induced to differentiation. Splicing products were analyzed for exon 16 inclusion by RT-PCR. +E16 indicates spliced products with exon 16; −E16, spliced products without exon 16; and +E16 (%), the percentage of spliced products includes exon 16. For each construct, 2 transfections were performed per experiment. Each experiment was repeated at least 3 times. The results shown are from 3 independent transfections. (C) Structure of the DUP minigene. DUP exon 1 is β-globin exon 1, and exon 3 is β-globin exon 2. The diagonal line in the second DUP exon indicates a fusion between the first and second β-globin exons to make a 33-nucleotide hybrid exon (E2). 3 copies of the wild-type (3WT) or mutant (3MU) sequences were inserted into the BglII site. (D) Splicing of DUP, DUP-3WT, and DUP-3MU in HeLa as well as in uninduced and induced MELCs. HeLa and MELCs were stably transfected with the vector alone (DUP), with 3 copies of the wild-type DUP-3WT, or with 3 copies of the mutated DUP-3MU. MELC stable lines were induced to differentiation with DMSO for 4 days. RNA were isolated and analyzed for E2 expression by RT-PCR. +E2 indicates spliced products with exon E2; −E2, spliced products without exon E2; and +E2(%), percentage of spliced products that include exon E2.