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J. Biol. Chem., Vol. 269, Issue 17, 12488-12493, 04, 1994

Exclusion of an alternatively spliced exon in human ATP synthase gamma- subunit pre-mRNA requires de novo protein synthesis

H Endo, C Matsuda and Y Kagawa
Department of Biochemistry, Jichi Medical School, Tochigi, Japan.

Tissue-specific isoforms of the mitochondrial ATP synthase gamma- subunit are generated by alternative splicing, and the heart/skeletal muscle-specific transcript lacks exon 9 in a cassette fashion (Matsuda, C., Endo, H., Hirata, H., Morosawa, H., Nakanishi, M., and Kagawa, Y. (1993) FEBS Lett. 325, 281-284; and Matsuda, C., Endo, H., Ohta, S., and Kagawa, Y. (1993) J. Biol. Chem. 268, 24950-24958). Here, we show that the endogenous heart-type mRNA is cell-specifically induced by the extracellular pH value in the HT1080 (human fibrosarcoma) and KYM-1 (human rhabdomyosarcoma) cell lines. In these cells, a low extracellular pH value induced exclusion of exon 9, and this induction was inhibited by cycloheximide treatment. In contrast, a high extracellular pH value resulted in mRNA transcription of the liver type, including exon 9, and did not require de novo protein synthesis. These results suggest that alternative splicing in the gamma-subunit pre-mRNA is regulated by on-off switching of protein synthesis of a trans-acting factor involved in this exon-excluding step. The signal of low pH value was blocked by the protein kinase inhibitor H-7 or Calphostin C (protein kinase C inhibitor), indicating the involvement of protein kinase C in the alternative splicing. This is a good model system for studies on the induction mechanism of alternative splicing in cultured mammalian cells, in which intracellular factors play a pivotal role for the exon-excluding step in the tissue-specific alternative splicing mechanism.
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