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. 2004 Dec;24(24):10766-76.
doi: 10.1128/MCB.24.24.10766-10776.2004.

5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs

Feng Fang  1 , Jason Hoskins, J Scott Butler
Affiliations

5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs

Feng Fang et al. Mol Cell Biol. 2004 Dec.

Abstract

The antimetabolite 5-fluorouracil (5FU) is a widely used chemotherapeutic for the treatment of solid tumors. Although 5FU slows DNA synthesis by inhibiting the ability of thymidylate synthetase to produce dTMP, the drug also has significant effects on RNA metabolism. Recent genome-wide assays for 5FU-induced haploinsufficiency in Saccharomyces cerevisiae identified genes encoding components of the RNA processing exosome as potential targets of the drug. In this report, we used DNA microarrays to analyze the effect of 5FU on the yeast transcriptome and found that the drug causes the accumulation of polyadenylated fragments of the 27S rRNA precursor and that defects in the nuclear exoribonuclease Rrp6p enhance this effect. The size distribution of these RNAs and their sensitivity to Rrp6p suggest that they are normally degraded by the nuclear exosome and a 5'-3' exoribonuclease. Consistent with this hypothesis, 5FU inhibits the growth of RRP6 mutants with defects in the degradation function of the enzyme and it interferes with the degradation of an rRNA precursor. The detection of poly(A)(+) pre-RNAs in strains defective in various steps in ribosome biogenesis suggests that the production of poly(A)(+) pre-rRNAs may be a general result of defects in rRNA processing. These findings suggest that 5FU inhibits an exosome-dependent surveillance pathway that degrades polyadenylated precursor rRNAs.

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Figures

FIG. 1.
FIG. 1.
5FU-induced growth deficiency in various yeast strains. Yeast strains were grown to an A600 of 0.5 to 1.0, and 10-fold serial dilutions were spotted onto synthetic complete plates containing 2% dextrose (SCD) in the absence or presence of 2 μM 5FU. The plates were incubated for 2 days at 30°C. All diploid strains contained Kanr knockouts of the indicated genes in the background of the normal (+/+) strain YSB2001. All haploid strains contained Kanr knockouts of the indicated genes in the background of the normal (RRP6) strain YSB1002.
FIG. 2.
FIG. 2.
5FU causes the accumulation of poly(A)+ rRNAs. (A) Fold change in signal as a function of the microarray oligonucleotide probe set specific for the rRNA locus from each of the indicated strains grown in YPD at 30°C and treated or untreated with 20 μM 5FU for 60 min. The diagram below the chart represents the rRNA locus. The lines above represent the positions and lengths of sequence coverage of individual probe sets on the Affymetrix S98 microarray chips. The arrows point to positions in the chart where the fold changes from the respective probe sets are illustrated. (B) RT-PCR analysis of the levels of 27S rRNA from the indicated strains. Total RNA from each of the strains was reverse transcribed using an oligo(dT) primer, followed by PCR for 20 cycles using oligo(dT) and OSB402 as primers. M, molecular weight markers.
FIG. 3.
FIG. 3.
Ribosomal RNAs from 5FU-treated strains bind to oligo(dT)-cellulose. (A) Simplified diagram of the processing of 35S pre-rRNA into 32S, 27S, and 7S pre-rRNAs. The diagram indicates the position to which the indicated oligonucleotide probes bind 27S pre-rRNA. T21, sequence of 21 T's following the 3′ end of OSB380. (B to D) Northern blot analysis of oligo(dT)-selected RNAs. Total RNA from the indicated strains untreated or treated with 20 μM 5FU for 60 min was separated into unbound [poly(A)− (A−)] and bound [poly(A)+ (A+)] fractions and analyzed by Northern blotting with the indicated radiolabeled oligonucleotide probes. Lanes 13 to 18 are shown once at the same exposure as that of lanes 1 to 12 (left) and again at a longer exposure (right panel).
FIG. 4.
FIG. 4.
Ribosomal processing mutations cause accumulation of poly(A)+ 27S rRNAs. (A) RNA titration of the RT-PCR assay mixture. Reverse transcription with an oligo(dT) primer was carried out with twofold dilutions of DNase-treated total RNA from an rrp6−/− strain, followed by PCR using primers OSB451 and OSB452 for ACT1 mRNA and OSB402 and OSB379 for 27S rRNA. (B) RT-PCR analysis of DNase-treated total RNA from the indicated strains. The top panel shows 2% agarose gel electrophoretic analysis of the RT-PCR products. The bottom panel shows a Northern blot of the same RNA samples. The blot was hybridized with OSB157. Strains producing RNA in lanes 1 to 14 were grown in SCD at 30°C and treated with 10 μg of doxycycline/ml for 24 h before RNA isolation.
FIG. 5.
FIG. 5.
5FU inhibits degradation of rRNA. (A) Tenfold serial dilutions of strains with the indicated genotypes were incubated at 30°C on SCD-URA-MET plates with or without the indicated amount of 5FU. (B) Northern blot analysis of RNA from the indicated strains grown as described for panel A with (+) or without (−) 20 μM 5FU. The blot in the top panel was probed with OSB155 and the blot in the bottom panel was probed with OSB151 for SCR1 RNA. The positions of A2-C2 RNA and SCR1 RNA are indicated to the left of the panels.
FIG. 6.
FIG. 6.
5FU inhibits 60S ribosome formation. Strains with the indicated genotypes were grown in the presence or absence (−) of 20 μM 5FU at 30°C, and cell extracts were prepared in the presence of cycloheximide. Extracts were separated by ultracentrifugation through 15 to 50% sucrose gradients, and the positions of ribosomes were determined by continuous analysis of the absorbance at 254 nM and are drawn in the figure using the same scale. The positions of the polyribosomes, the 80S, 60S, and 40S ribosome subunits, and the 60S/40S ratio are indicated in each panel.
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