STUDY FOR MOLECULAR MECHANISM OF MUSCLE-SPECIFIC ALTERNATIVE RNA SPLICING.

线粒体三磷酸腺苷合成酶提供真核细胞中大部分三磷酸腺苷。肌特异性三磷酸腺苷合成酶γ亚基亚基(F_1γ)是通过组织特异性选择性剪接产生的。在本研究中，我们研究了肌肉特异性选择性剪接在F_1期γ前基因中的分子机制。首先，我们利用人纤维肉瘤(HT1080)细胞建立了肌肉特异性选择性剪接的可逆诱导系统，并利用肌肉特异性剪接诱导的HT1080细胞的核提取液构建了反映人F_1γ微型基因肌肉特异性选择性剪接的体外剪接系统。通过对F_1γ微型基因的体外突变分析，我们在一个交替剪接的外显子上发现了一个外显子剪接增强子(Ese)和一个肌肉特异性外显子剪接沉默(MS-Ess)。ESE是构成外显子选择所必需的，MS-ESS在肌肉特异性剪接条件下特异性地起到排除外显子的作用。利用这些顺式调控元件进行UV交联和North-Western印迹分析，发现一个48 kDa的蛋白和一个42 kDa的蛋白是RNA结合蛋白，分别与ESE和ME-ESS结合。最后，我们还利用携带F_1γ微型基因的转基因小鼠描述了多管肌纤维和成熟肌纤维之间肌肉特异性剪接调控机制的差异。必须确定这些剪接调控因子，才能了解肌肉特异性选择性剪接的分子机制。

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.