Both the RRM and the C-terminal domain are required for exon repression when tethered to the upstream intron. (A) Diagrams of the modified β-globin-PB1 minigene and MS2-fused RBFOX1 constructs. Three natural RBFOX1/2 binding sites in the upstream intron and one natural binding site in the alternative exon were mutated, and an MS2-binding site was inserted in the upstream intron. The primers for RT–PCR are indicated by arrows below the exons. All of the protein mutants have a Flag epitope tag, which is indicated by an open circle. The RRM with two Phe-to-Asp mutations is designated RRMDD. (B) HeLa cells were cotransfected with the β-globin-PB1-MS2up reporter minigene and different RBFOX1 constructs. RNA and protein were extracted 48 h after transfection. Radioactive RT–PCR was performed to detect the changes of the alternative splicing isoforms. (C) Western blot analysis using Flag antibody was performed to show the expression of the protein mutants.
