text: Effect of 5FU in tRNA transcripts and in the viability | Open-i
  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).