Figure 5.
Fox-1/2-mediated splicing regulation depends on the UGCAUG elements. (A) Schematic representation of the FMNL3 minigene, which has four natural copies of putative Fox-1/2-binding sites (labeled 1–4) in DIF sequences. Different use of stop codons due to AS is also indicated by red circles. The conservation pattern of the region is displayed under the diagram. (B) Splicing of the FMNL3 minigene cassette exon in the wild-type minigene, without or with Fox-1 protein, is shown in lanes 1 and 2, respectively. Lanes 3–8 show the splicing of the mutant minigenes. Mut12 (lanes 3,4) has mutations in sites 1 and 2, and similarly for Mut34 (lanes 5,6) and Mut 1234 (lanes 7,8). The quantified exon inclusion level is indicated. The expression level of Fox-1 was confirmed by Western blotting, as shown at the bottom. (C) Schematic representation of the PB1 minigene. The conservation pattern of the region is displayed under the diagram. The cassette exon, together with ∼250 nt of UIF and DIF sequences, including three natural putative Fox-1/2-binding sites in the UIF region, were inserted into intron 1 of the human β-globin gene. (D) Splicing of the PB1 minigene. See the legend for B for more details.