RNA-Based 5-Fluorouracil Toxicity Requires the Pseudouridylation Activity of Cbf5p

Abstract

The chemotherapeutic drug 5-fluorouracil (5FU) disrupts DNA synthesis by inhibiting the enzymatic conversion of dUMP to dTMP. However, mounting evidence indicates that 5FU has important effects on RNA metabolism that contribute significantly to the toxicity of the drug. Strains with mutations in nuclear RNA-processing exosome components, including Rrp6p, exhibit strong 5FU hypersensitivity. Studies also suggest that 5FU-containing RNA can inhibit pseudouridylation, the most abundant post-transcriptional modification of noncoding RNA. We examined the effect of modulating the expression and activity of the essential yeast rRNA pseudouridylase Cbf5p on the 5FU hypersensitivity of an rrp6-D mutant strain. Depletion of Cbf5p suppressed the 5FU hypersensitivity of an rrp6-D strain, while high-copy expression enhanced sensitivity to the drug. A mutation in the catalytic site of Cbf5p also suppressed the 5FU hypersensitivity in the rrp6-D mutant, suggesting that RNA-based 5FU toxicity requires the pseudouridylation activity of Cbf5p. High-copy expression of box H/ACA snoRNAs also suppressed the 5FU hypersensitivity of an rrp6-D strain, suggesting that sequestration of Cbf5p to a particular guide RNA reduces Cbf5p-dependent 5FU toxicity. On the basis of these results and previous reports that certain pseudouridylases form stable adducts with 5FU-containing RNA, we suggest that Cbf5p binds tightly to substrates containing 5FU, causing their degradation by the TRAMP/exosome-mediated RNA surveillance pathway.

The Incorporation of 5-Fluorouracil into RNA Affects the Ribonucleolytic Activity of the Exosome Subunit Rrp6

“…The molecular mass of the nuclear exosome is altered, which suggests that its composition or its association with other factors is modified. This observation is consistent with the suggestion that incorporation of 5FU into RNA leads to sequestration of Rrp6 (7,18). Second, the ribonucleolytic activity of Rrp6 is less efficient on RNA that contains 5FU than it is on normal RNA.…”

“…In a recent study (7), Hoskins and Butler have implicated the activity of the yeast rRNA pseudouridylase Cbf5p in the cytotoxicity of 5FU, and have suggested that Cbf5p forms stable adducts with 5F-RNA in cells treated with 5FU. The toxic effect of 5FU may thus be partly mediated by the sequestration of pseudouridylases in inactive ribonucleoprotein complexes (7). In such a scenario, psuedouridylation reactions would be inhibited and the 5F-RNA bound to pseudouridylases would be degraded by Rrp6 and the nuclear exosome.…”

“…The results presented here together with results of other authors establish that 5FU affects RNA metabolism in multiple ways. On the one hand, the incorporation of 5FU into RNA blocks the processing of RNA precursors, including pre-mRNAs and rRNAs (4,5,7), which in itself is detrimental for RNA biogenesis. Moreover, 5F-RNA sequesters pseudouridylases in stable ribonucleoprotein complexes, which further limits the capacity of the cell to modify RNA precursors (7).…”

“…On the one hand, the incorporation of 5FU into RNA blocks the processing of RNA precursors, including pre-mRNAs and rRNAs (4,5,7), which in itself is detrimental for RNA biogenesis. Moreover, 5F-RNA sequesters pseudouridylases in stable ribonucleoprotein complexes, which further limits the capacity of the cell to modify RNA precursors (7). On the other hand, 5FU makes RNA less susceptible to degradation by the surveillance machinery, as shown here, and reduces the cellular levels of Rrp6 (18), a consequence of which is that aberrant transcripts synthesized by 5FU-treated cells cannot be turned over efficiently.…”

“…Excision of the misincorporated 5FU derivatives by uracil-DNA-glycosylase results in DNA damage and cell death (3). And third, FUTP is extensively incorporated into RNA, which inhibits the processing and maturation of rRNA, tRNA, and mRNA precursors (4)(5)(6)(7).…”