Abstract
A quantitative polymerase chain reaction (PCR) assay has been developed to determine the absolute and relative amounts of each dihydrofolate reductase RNA species present at different stages of splicing (i.e., pre-mRNA, splicing intermediate, and mRNA). The ratios of each RNA species as measured by quantitative PCR have been confirmed by S1 nuclease mapping analysis. Quantitative PCR studies reveal a concentration-dependent decrease in the levels of dihydrofolate reductase mRNA and a splicing intermediate but little change in pre-mRNA levels after long term exposure of cells to 5-fluorouracil (FUra). The observed changes correlate with the extent of FUra incorporation into RNA and with cytotoxicity. These results, together with previous data from our laboratory, provide the first direct evidence that FUra incorporation into RNA can cause inhibition of pre-mRNA splicing in vivo. Inhibition of pre-mRNA splicing is thus a likely additional mechanism by which FUra incorporation into RNA may lead to growth inhibition and cell death.
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