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New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

Mojardín L, Botet J, Quintales L, Moreno S, Salas M - PLoS ONE (2013)

Bottom Line: This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment.The transcription of several tRNA genes was also significantly induced after drug exposure.Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, Madrid, Spain.

ABSTRACT
5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

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Related in: MedlinePlus

Box and whisker plots showing the ratio of intron/exon hybridization signal intensities after 5FU exposure.The analysis considered 948 intron-containing transcripts of S. pombe. The ratio represents the pre-mRNA/(mRNA+pre-mRNA) levels. The Individual boxes represent the median (central horizontal line) and the 75–25% percentiles. The whiskers extend from the boxes to 1% and 99% of the data set. Dots indicate outliers. Data are representative of two independent experiments. The P values between groups were calculated using the two-tailed Mann-Whitney test. The values at 240 min for introns SPCC24B10.17_b (5.2) and SPAC1486.01_a (4.9) are not shown.
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pone-0078172-g002: Box and whisker plots showing the ratio of intron/exon hybridization signal intensities after 5FU exposure.The analysis considered 948 intron-containing transcripts of S. pombe. The ratio represents the pre-mRNA/(mRNA+pre-mRNA) levels. The Individual boxes represent the median (central horizontal line) and the 75–25% percentiles. The whiskers extend from the boxes to 1% and 99% of the data set. Dots indicate outliers. Data are representative of two independent experiments. The P values between groups were calculated using the two-tailed Mann-Whitney test. The values at 240 min for introns SPCC24B10.17_b (5.2) and SPAC1486.01_a (4.9) are not shown.

Mentions: The fission yeast nuclear genome consists of 5,175 annotated protein-coding genes, of which 2,404 are known or predicted to contain at least one intron with an average length of 81 nucleotides [20]. To obtain a direct measurement of intron-containing transcripts we calculated the average probe intensity across the 948 intronic regions that were delimited by, at least, 4 core probes in the microarray platform, since those data were deemed to be statistically significant (Table S3). Cells treated with 5FU showed a time-dependent global increase of unspliced transcripts, whereas the exon levels (representing a measure of both mature mRNA and pre-mRNA levels) did not significantly change, ruling out the possibility that these alterations were an indirect consequence of increments in the transcription rates (Figure S2). Consistent with this result is the fact that the levels of intronic regions relative to their corresponding flanking exons (intron/exon ratio) were significantly higher at 60 and 240 min after drug exposure than the untreated control (P<0.05 and P<0.0001, respectively) (Figure 2). In particular, more than 1.5-fold increase was detected for 12% of the genes at 60 min and 27.7% at 240 min of 5FU treatment (Table S3).


New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

Mojardín L, Botet J, Quintales L, Moreno S, Salas M - PLoS ONE (2013)

Box and whisker plots showing the ratio of intron/exon hybridization signal intensities after 5FU exposure.The analysis considered 948 intron-containing transcripts of S. pombe. The ratio represents the pre-mRNA/(mRNA+pre-mRNA) levels. The Individual boxes represent the median (central horizontal line) and the 75–25% percentiles. The whiskers extend from the boxes to 1% and 99% of the data set. Dots indicate outliers. Data are representative of two independent experiments. The P values between groups were calculated using the two-tailed Mann-Whitney test. The values at 240 min for introns SPCC24B10.17_b (5.2) and SPAC1486.01_a (4.9) are not shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815194&req=5

pone-0078172-g002: Box and whisker plots showing the ratio of intron/exon hybridization signal intensities after 5FU exposure.The analysis considered 948 intron-containing transcripts of S. pombe. The ratio represents the pre-mRNA/(mRNA+pre-mRNA) levels. The Individual boxes represent the median (central horizontal line) and the 75–25% percentiles. The whiskers extend from the boxes to 1% and 99% of the data set. Dots indicate outliers. Data are representative of two independent experiments. The P values between groups were calculated using the two-tailed Mann-Whitney test. The values at 240 min for introns SPCC24B10.17_b (5.2) and SPAC1486.01_a (4.9) are not shown.
Mentions: The fission yeast nuclear genome consists of 5,175 annotated protein-coding genes, of which 2,404 are known or predicted to contain at least one intron with an average length of 81 nucleotides [20]. To obtain a direct measurement of intron-containing transcripts we calculated the average probe intensity across the 948 intronic regions that were delimited by, at least, 4 core probes in the microarray platform, since those data were deemed to be statistically significant (Table S3). Cells treated with 5FU showed a time-dependent global increase of unspliced transcripts, whereas the exon levels (representing a measure of both mature mRNA and pre-mRNA levels) did not significantly change, ruling out the possibility that these alterations were an indirect consequence of increments in the transcription rates (Figure S2). Consistent with this result is the fact that the levels of intronic regions relative to their corresponding flanking exons (intron/exon ratio) were significantly higher at 60 and 240 min after drug exposure than the untreated control (P<0.05 and P<0.0001, respectively) (Figure 2). In particular, more than 1.5-fold increase was detected for 12% of the genes at 60 min and 27.7% at 240 min of 5FU treatment (Table S3).

Bottom Line: This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment.The transcription of several tRNA genes was also significantly induced after drug exposure.Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, Madrid, Spain.

ABSTRACT
5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.

Show MeSH
Related in: MedlinePlus