The C-terminal domain of RBFOX1 is sufficient for exon activation when tethered to the downstream intron. (A) Diagrams of the modified SMN2 minigene and MS2-fused RBFOX1 constructs. An MS2 binding site was inserted in the intron downstream from exon 7. The primers for RT–PCR are indicated by arrows below the exons. RBFOX1 has one RRM (gray) flanked by N-terminal (black square) and C-terminal (white oval) domains. All of the protein constructs have a Flag epitope tag at the N terminus, indicated by an open circle. The MS2 module also has a T7 tag at its N terminus, represented by a vertical bar. (B) HeLa cells were cotransfected with the SMN2-MS2down reporter minigene and different protein constructs. RNA and proteins were extracted 48 h after transfection. Radioactive RT–PCR was performed to measure the changes in the alternative splicing isoforms. (C) Western blot analysis using Flag antibody was performed to show the expression of the protein mutants. (*) Nonspecific band.
