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Screening system for chemicals attacking pre-mRNA splicing machinery in cells

攻击细胞内前 mRNA 剪接机制的化学物质筛选系统

基本信息

批准号：
13670334
负责人：
NISHIO Hisahide
金额：
$ 2.24万
依托单位：
Kobe University Graduate School of medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670334/
关键词：
pre-mRNA splicing splicing enhancer sequences the dsx gene mini-gene anti-SMN antibody spinal muscular atrophy cadmium メッセンジャーRNA前駆体 SMN蛋白可変スプライシング

项目摘要

It has been expected that some environmental chemicals attack the cellular splicing machinery to block the gene functions. Such chemicals, however, have never been reported so far. During our investigation of some inherited disorders with abnormal splicing pattern of the genes, we established splicing assay system with artificial pre-mRNA. In 2001, we constructed a mini-gene, of which transcripts were used as pre-mRNA. This mini-gene was a chimeric plasmid which consisted of T7 promoter, the third exon-the third intron-the 5' part of the fourth exon of the Drosophila melanogaster double sex gene (dsx) and splicing enhancer sequences (SES) selected from exons of the human dystrophin gene.In 2002, we performed splicing assays using the mini-genes in the HeLa nuclear extract. High splicing efficiency was observed in the assays using the mini-genes with SESs from exons 19 and 26, When anti-SMN antibody was added in the HeLa nuclear extract, splicing reaction using the mini-gene with SES fr … More om exon 19 was blocked but splicing reaction using the mini-gene with SES from exon 26 was not blocked. The anti-SMN antibody is against the SMN protein, which is a product of the responsible gene for spinal muscular atrophy. when cadmium chloride (CdC12) solution was added in the HeLa nuclear extract, splicing reaction using the mini-gene with SES from exon 19 was blocked. The degree of splicing blockade was dependent on the CdC12 concentration.These findings suggest that splicing efficiency is, at least partly, dependent on the SESs ; some chemical may affect splicing of some exons, but the same chemical may not affect splicing of the other exons. We can also expect that some chemical may affect splicing of some exons in the HeLa nuclear extract, but the same chemical may not affect splicing of the same exons in the non-HeLa nuclear extract. However, our preliminary study showed a new kind of cadmium toxicity affecting splicing machinery. In addition, the present study with anti-SMN antibody gives some clues to understanding of the pathogenesis of spinal muscular atrophy. Less

人们预计某些环境化学物质会攻击细胞剪接机制以阻断基因功能。然而，迄今为止，此类化学物质从未被报道过。在我们对一些基因剪接模式异常的遗传性疾病的研究过程中，我们建立了人工前体mRNA剪接检测系统。 2001年，我们构建了一个小基因，其转录物被用作前mRNA。该小基因是由黑腹果蝇双性基因(dsx)的T7启动子、第三外显子-第三内含子-第四外显子5'部分和选自人抗肌营养不良蛋白基因外显子的剪接增强子序列(SES)组成的嵌合质粒。2002年，我们利用HeLa核提取物中的小基因进行了剪接实验。在使用带有来自外显子 19 和 26 的 SES 的小基因的测定中观察到高剪接效率，当在 HeLa 核提取物中添加抗 SMN 抗体时，使用带有来自外显子 19 的 SES 的小基因的剪接反应被阻断，但使用带有来自外显子 26 的 SES 的小基因的剪接反应没有被阻断。抗 SMN 抗体针对 SMN 蛋白，该蛋白是脊髓性肌萎缩症相关基因的产物。当在 HeLa 核提取物中添加氯化镉 (CdC12) 溶液时，使用小基因与外显子 19 的 SES 进行的剪接反应被阻断。剪接阻断程度取决于 CdC12 浓度。这些发现表明剪接效率至少部分取决于 SES；某些化学物质可能会影响某些外显子的剪接，但相同的化学物质可能不会影响其他外显子的剪接。我们还可以预期，某些化学物质可能会影响 HeLa 核提取物中某些外显子的剪接，但相同的化学物质可能不会影响非 HeLa 核提取物中相同外显子的剪接。然而，我们的初步研究表明，一种新型镉毒性会影响熔接机械。此外，目前抗SMN抗体的研究为了解脊髓性肌萎缩症的发病机制提供了一些线索。较少的