Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants.(A) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNAAlaCGC, tRNAArgCCU and tRNAGlyCCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P<0.05, ** P<0.005, using the two-tailed Student’s t test. T represents the amount of total tRNA (combining both spliced and unspliced transcripts), whereas I represents the intron levels. (B) Sensitivity to 5FU of S. pombe strains deleted for genes involved in the modification of residues within tRNA molecules. The growth of the 5FU sensitive strain dus3Δ is also shown. Note that the control wild-type is the ED668 strain. The growth curve is representative of at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815194&req=5

pone-0078172-g004: Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants.(A) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNAAlaCGC, tRNAArgCCU and tRNAGlyCCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P<0.05, ** P<0.005, using the two-tailed Student’s t test. T represents the amount of total tRNA (combining both spliced and unspliced transcripts), whereas I represents the intron levels. (B) Sensitivity to 5FU of S. pombe strains deleted for genes involved in the modification of residues within tRNA molecules. The growth of the 5FU sensitive strain dus3Δ is also shown. Note that the control wild-type is the ED668 strain. The growth curve is representative of at least two independent experiments.

Mentions: Intron-containing transcripts from protein-coding genes are not the only type of RNA molecules that undergo splicing in eukaryotic cells. A number of tRNA genes also contain introns that must be removed by an enzymatic cut-and-rejoin reaction that requires the heterotetrameric tRNA splicing endonuclease (SEN) complex to generate the mature and functional structure. Using the tRNAscan-SE program (Genomic tRNA Database, http://gtrnadb.ucsc.edu) a total of 186 tRNA genes were identified in S. pombe, 44 of which have introns (24% of the total) ranging in size from 7 to 30 nucleotides [25]. To test whether the pre-tRNA splicing was affected by 5FU, we compared the levels of unspliced transcripts after 240 min of drug exposure respect to the untreated control for four selected intron-containing tRNA genes that code for tRNAAlaCGC, tRNAArgCCU, tRNALeuCAA and tRNASerGCU (Figure 4A). Specific intronic or exonic regions within each tRNA were amplified using reverse transcription and qPCR experiments. A comparison of the intron to exon ratios at 0 and 240 min of 5FU treatment for each type of tRNA transcript revealed that only tRNAArgCCU showed a statistically significant increase in intron retention (1.2-fold higher, P = 0.05). The results also indicated that cell exposure to 5FU for 240 min had an enhanced expression of the selected intron-containing tRNA genes (Figure 4A). This finding led us to ask whether the levels of non intron-containing tRNA were also affected. The analysis showed that the amount of transcripts detected for tRNAHisGUG, tRNATrpCCA, tRNAGlyCCC, tRNAValUAC and tRNAGluCUC were notably higher after 5FU exposure, although their levels were lower than those in most of the intron-containing tRNA genes examined (Figure 4A).


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)

Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants.(A) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNAAlaCGC, tRNAArgCCU and tRNAGlyCCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P<0.05, ** P<0.005, using the two-tailed Student’s t test. T represents the amount of total tRNA (combining both spliced and unspliced transcripts), whereas I represents the intron levels. (B) Sensitivity to 5FU of S. pombe strains deleted for genes involved in the modification of residues within tRNA molecules. The growth of the 5FU sensitive strain dus3Δ is also shown. Note that the control wild-type is the ED668 strain. The growth curve is representative of at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078172-g004: Effect of 5FU in tRNA transcripts and in the viability of different tRNA modification mutants.(A) Relative levels of intronic or exonic regions within transcripts of 9 selected tRNA genes at both 0 and 240 min of 5FU exposure quantified by qPCR. Genes for tRNAAlaCGC, tRNAArgCCU and tRNAGlyCCC only have one copy in the genome, whereas multiple identical copies are found for the other tRNA genes examined. In these cases, the amplified products detected represent a mixture of the expression levels of the individual repetitions. The values represent the relative fold change in transcripts abundance between untreated and 5FU-treated cells normalized to the initial amount of starting RNA. Note that only differences between the same group are directly comparable. The expression levels of the normalization gene myo1 were shown as a control. Bar charts show the average values ± s.d. of two independent experiments. Asterisks designate overall significance, * P<0.05, ** P<0.005, using the two-tailed Student’s t test. T represents the amount of total tRNA (combining both spliced and unspliced transcripts), whereas I represents the intron levels. (B) Sensitivity to 5FU of S. pombe strains deleted for genes involved in the modification of residues within tRNA molecules. The growth of the 5FU sensitive strain dus3Δ is also shown. Note that the control wild-type is the ED668 strain. The growth curve is representative of at least two independent experiments.
Mentions: Intron-containing transcripts from protein-coding genes are not the only type of RNA molecules that undergo splicing in eukaryotic cells. A number of tRNA genes also contain introns that must be removed by an enzymatic cut-and-rejoin reaction that requires the heterotetrameric tRNA splicing endonuclease (SEN) complex to generate the mature and functional structure. Using the tRNAscan-SE program (Genomic tRNA Database, http://gtrnadb.ucsc.edu) a total of 186 tRNA genes were identified in S. pombe, 44 of which have introns (24% of the total) ranging in size from 7 to 30 nucleotides [25]. To test whether the pre-tRNA splicing was affected by 5FU, we compared the levels of unspliced transcripts after 240 min of drug exposure respect to the untreated control for four selected intron-containing tRNA genes that code for tRNAAlaCGC, tRNAArgCCU, tRNALeuCAA and tRNASerGCU (Figure 4A). Specific intronic or exonic regions within each tRNA were amplified using reverse transcription and qPCR experiments. A comparison of the intron to exon ratios at 0 and 240 min of 5FU treatment for each type of tRNA transcript revealed that only tRNAArgCCU showed a statistically significant increase in intron retention (1.2-fold higher, P = 0.05). The results also indicated that cell exposure to 5FU for 240 min had an enhanced expression of the selected intron-containing tRNA genes (Figure 4A). This finding led us to ask whether the levels of non intron-containing tRNA were also affected. The analysis showed that the amount of transcripts detected for tRNAHisGUG, tRNATrpCCA, tRNAGlyCCC, tRNAValUAC and tRNAGluCUC were notably higher after 5FU exposure, although their levels were lower than those in most of the intron-containing tRNA genes examined (Figure 4A).

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