| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
Mitochondrial ATP synthase provides most ATP in eukaryotic cells. The muscle-specific isoform of ATP synthase γ-subunit (F_1γ) is generated by tissue-specific alternative splicing. In this study, we investigated on the molecular mechanism of muscle-specific alternative splicing in F_1γ pre-mRNA. First, we developed the reversible induction system for muscle-specific alternative splicing using human fibrosarcoma (HT1080) cells, and also constructed an in vitro splicing system reflecting muscle-specific alternative splicing in human F_1γ minigene, by addition with nuclear extracts from muscle-specific splicing-induced HT1080 cells. From mutation analysis of F_1γ minigene using this in vitro system, we identified an Exonic Splicing Enhancer (ESE) and a Muscle-specific Exonic Splicing silencer (MS-ESS) on an alternatively spliced exon. The ESE is required for constitutive exon selection, and the MS-ESS specifically acts for exon exclusion under the muscle-specific splicing condition. From UV cross-linking and North-Western blot analyses using these cis-regulatory elements, a 48 kDa protein and a 42 kDa protein were detected as RNA-binding proteins, which bind to ESE and ME-ESS, respectively. Finally, we also described the difference of muscle-specific splicing reguratory mechanism between muotubes and mature muscle fibers using transgenic mice bearing F_1γ minigene. These splicing reguratory factors must be identified to understand the molecular mechanism of muscle-specific alternative splicing.
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