Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo

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Abstract
5-fluorouracil (5FU) is an effective anti-cancer drug, yet its mechanism of action remains unclear. Here, we examine the effect of 5FU on pre-mRNA splicing in vivo . Using RT–PCR, we show that the splicing of a number of pre-mRNAs is inhibited in HeLa cells that have been exposed to a low dose of 5FU. It appears that this inhibitory effect is not due to its incorporation into pre-mRNA, because partially or fully 5FU-substituted pre-mRNA, when injected into Xenopus oocytes, is spliced just as well as is the unsubstituted pre-mRNA. Detailed analyses of 5FU-treated cells indicate that 5FU is incorporated into U2 snRNA at important naturally occurring pseudouridylation sites. Remarkably, 5FU incorporation effectively blocks the formation of important pseudouridines in U2 snRNA, as only a trace of pseudouridine is detected when cells are exposed to a low dose of 5FU for 5 days. Injection of the hypopseudouridylated HeLa U2 snRNA into U2-depleted Xenopus oocytes fails to reconstitute pre-mRNA splicing, whereas control U2 isolated from untreated or uracil-treated HeLa cells completely reconstitutes the splicing. Our results demonstrate for the first time that 5FU incorporates into a spliceosomal snRNA at natural pseudouridylation sites in vivo , thereby inhibiting snRNA pseudouridylation and splicing. This mechanism may contribute substantially to 5FU-mediated cell death.