A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10
A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10
复制标题
DOI：
10.1111/j.1471-4159.2004.02477.x
复制
发表时间：
2004-07
影响因子：
4.7
通讯作者：
Qingming Yu;Jun Guo;Jianhua Zhou
中科院分区：
文献类型：
--
作者：
Qingming Yu;Jun Guo;Jianhua Zhou
刺激外显子10包含到人类tau mRNA中的突变导致额颞叶痴呆伴帕金森综合征，与17号染色体（FTDP-17）和其他tau蛋白病相关。这表明成年脑中外显子10包含与排除的比例是决定tau蛋白生物学功能的因素之一。为了研究潜在的剪接机制并确定tau蛋白病的潜在治疗靶点，我们从全长tau外显子9-11小基因构建体中产生了一系列内含子缺失的小基因构建体。RT-PCR结果表明，外显子10和11之间存在正确剪接外显子10的最小距离要求。此外，发现剪接因子的丝氨酸-精氨酸（SR）蛋白家族成员SRp 20以剂量依赖性方式促进外显子10的排除。值得注意的是，SRp 20还诱导了FTDP-17患者中含有突变的前mRNA的外显子10跳跃。基于这些结果，我们生成了一个基于细胞的系统，使用荧光素酶报告基因测量tau mRNA中外显子10的包含和排除。萤火虫荧光素酶在读框中融合到外显子11中，并且在外显子10中也产生终止码。包含外显子10阻止荧光素酶表达，而排除外显子10产生荧光素酶活性。为了最大限度地减少基线荧光素酶表达，我们的报告基因构建体还包含FTDP-17突变，该突变增加了外显子10的包含。我们证明，我们的报告构建体的剪接模式模仿内源性tau基因。SRp 20和SRp 55（两种促进外显子10排除的SR蛋白）的共转染增加了荧光素酶的产生。我们的结论是，这种基于细胞的系统可用于识别调节外显子10剪接的生物物质。
Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP‐17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini‐gene constructs with intron deletions from the full length of tau exons 9–11 mini‐gene construct. RT–PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine‐arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage‐dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre‐mRNAs containing mutations identified in FTDP‐17 patients. Based on those results, we generated a cell‐based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP‐17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co‐transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell‐based system can be used to identify biological substances that modulate exon 10 splicing.