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J. Biol. Chem., Vol. 275, Issue 21, 15992-16001, May 26, 2000

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Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis^*

Masaru Ichida^§, Yoji Hakamata^¶, Morisada Hayakawa, Eriko Ueno, Uichi Ikeda^§, Kazuyuki Shimada^§, Toshiro Hamamoto, Yasuo Kagawa, and Hitoshi Endo

From the Departments of  Biochemistry and ^§ Cardiology and ^¶ Laboratory of Experimental Medicine, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan

Muscle-specific isoform of the mitochondrial ATP synthase  subunit (F₁) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F₁ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F₁ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F₁ Pu-del and F₁ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F₁ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F₁ Pu-rich minigene mice revealed that the F₁ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F₁ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

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