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

肌肉特异性选择性 RNA 剪接的分子机制研究。

基本信息

批准号：
11670132
负责人：
ENDO Hitoshi
金额：
$ 1.15万
依托单位：
JICHI MEDICAL SCHOOL
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

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.

线粒体ATP合酶提供真核细胞中的大部分ATP。ATP合成酶γ亚基（F_1γ）是通过组织特异性选择性剪接产生的肌肉特异性异构体。本研究旨在探讨F_1γ前体mRNA的肌肉特异性选择性剪接的分子机制。首先，我们利用人纤维肉瘤（HT 1080）细胞建立了肌肉特异性可变剪接的可逆诱导系统，并通过加入肌肉特异性剪接诱导的HT 1080细胞核提取物，构建了反映人F_1γ小基因肌肉特异性可变剪接的体外剪接系统。利用该体外系统对F_1γ小基因进行突变分析，在一个选择性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子（MS-ESS）。ESE是组成性外显子选择所必需的，而MS-ESS在肌肉特异性剪接条件下特异性地起外显子排除作用。从UV交联和North-Western印迹分析使用这些顺式调节元件，48 kDa的蛋白质和42 kDa的蛋白质被检测为RNA结合蛋白，其分别结合ESE和ME-ESS。最后，我们还利用F_1γ小基因转基因小鼠，对muotubes与成熟肌纤维之间的肌肉特异性剪接调节机制进行了比较。这些剪接调控因子必须被识别，以了解肌肉特异性选择性剪接的分子机制。