Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis.
Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) 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 F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma 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 F(1)gamma Pu-del and F(1)gamma 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 F(1)gamma 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 F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma 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 F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.
Ichida
M
M
Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan.
Hakamata
Y
Y
Hayakawa
M
M
Ueno
E
E
Ikeda
U
U
Shimada
K
K
Hamamoto
T
T
Kagawa
Y
Y
Endo
H
H
D025181:ATP Synthetase Complexes; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000235:genetics*; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics*; D020033:Protein Isoforms; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
D003470:Culture Media; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D012322:RNA Precursors; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor)