Acidic Stimulation Induces a Negative Regulatory Factor That Affects Alternative Exon Selectionin Vitroin Human ATP Synthase γ-Subunit Pre-mRNA

Abstract

Tissue-specific alternative RNA splicing in human F1γ pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1γ gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1γ minigene containing parts of exon 8, intron 8, and exon 9 of the human F1γ gene and then analyzed a negative factor that inhibited inclusion of exon 9 via anin vitrosplicing assay using acid-stimulated HT1080 cell nuclear extract.In vitrosplicing of the F1γ minigene, similarly to the β-globin minigene used as a control, was observed in HeLa cell nuclear extract. Next, we performed supplemental experiments using HeLa and HT1080 cell nuclear extracts. The splicing reaction of the F1γ minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human β-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1γ minigenein vitro,and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1γ pre-mRNA.