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

人们一直认为，一些环境化学物质会攻击细胞剪接机制，从而阻断基因的功能。然而，迄今为止，此类化学物质从未被报道过。在对一些基因剪接模式异常的遗传性疾病的研究中，我们建立了人工pre-mRNA剪接检测系统。2001年，我们构建了一个迷你基因，其转录本被用作pre-mRNA。该迷你基因是由果蝇双性基因（dsx）的T7启动子、第3外显子-第3内含子-第4外显子的5'部分和从人类肌营养不良蛋白基因外显子中选择的剪接增强子序列（SES）组成的嵌合质粒。2002年，我们使用HeLa核提取物中的迷你基因进行剪接试验。在HeLa核抽提液中加入抗smn抗体后，第19外显子的剪切反应被阻断，而第26外显子的剪切反应未被阻断。抗SMN抗体是针对SMN蛋白的，SMN蛋白是脊髓性肌萎缩的负责基因的产物。在HeLa核提取液中加入氯化镉（CdC12）溶液后，具有19外显子SES的迷你基因的剪接反应被阻断。剪接阻断程度依赖于CdC12的浓度。这些发现表明，剪接效率至少部分取决于SESs；某些化学物质可能影响某些外显子的剪接，但相同的化学物质可能不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，我们的初步研究发现了一种新的镉毒性影响拼接机制。此外，目前的抗smn抗体研究为了解脊髓性肌萎缩的发病机制提供了一些线索。少

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