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

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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