Acidic stimulation induces a negative regulatory factor that affects alternative exon selection in vitro in human ATP synthase gamma-subunit pre-mRNA.
Tissue-specific alternative RNA splicing in human F1gamma pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1gamma gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1gamma minigene containing parts of exon 8, intron 8, and exon 9 of the human F1gamma gene and then analyzed a negative factor that inhibited inclusion of exon 9 via an in vitro splicing assay using acid-stimulated HT1080 cell nuclear extract. In vitro splicing of the F1gamma minigene, similarly to the beta-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 F1gamma minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human beta-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1gamma minigene in vitro, and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1gamma pre-mRNA.
Hayakawa
M
M
Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi, Kawachi-gun, 329-0498, Japan.
Endo
H
H
Hamamoto
T
T
Kagawa
Y
Y
D017398:Alternative Splicing; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D005091:Exons; D005354:Fibrosarcoma; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005813:Genes, Synthetic; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007438:Introns; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012208:Rhabdomyosarcoma; D014020:Tissue Extracts / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D012322:RNA Precursors; D014020:Tissue Extracts; D006180:Proton-Translocating ATPases