Introduction

Dph3 plays a role in diphthamide modification of eukaryotic translation elongation factor 2 (eEF2) and in Elongator mediated modifications of tRNAs1,2,3,4,5. Dph3 is an electron donor for Dph1-Dph2, which catalyses the first step of the eEF2 modification, the transfer of 3-amino-carboxypropyl from S-adenosylmethionine to a conserved histidine of eEF21, 5. In the second step, Dph5 tetra-methylates the 3-amino-carboxypropyl-histidine producing methylated diphthine. In the following step, catalysed by Dph7, de-methylation produces diphthine, which Dph6 converts to diphthamide in the final step6, 7. Modified eEF2 ensures error-free addition of amino acids to a growing peptide chain during translation by coordinating movement of tRNAs and mRNAs on ribosomes. In higher eukaryotes, mutations in DPH genes have been reported to be associated with embryonic lethality, neurological and developmental defects and cancer8,9,10,11,12,13,14.

tRNA wobble uridine modifications carried out by Elp3 as part of the Elongator complex require, like the diphthamide synthesis, S-adenosylmethionine as substrate, indicating that Dph3 also serves as an electron donor in this enzymatic reaction2,3,4. Elongator and accessory proteins catalyse 5-carbonylmethyl (cm5) on wobble uridines2, 15,16,17. Subsequently, cm5U34 of tRNALys UUU, tRNAGlu UUC, tRNAGln UUG, tRNAArg UCU and tRNAGly UCC is methylated to 5-methoxycarbonyl-methyluridine (mcm5U34) by Trm9-Trm11216, 18, 19. The wobble uridines of the first three tRNA species are also thiolated by the Urm1 pathway to form 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U34)20. cm5U34 in tRNALeu UUA, tRNAVal GUA, tRNASer UGA, tRNAPro UGG, tRNAThr UGU and tRNAAla UGC can be also modified to 5-carbamoyl-methyl-uridine (ncm5U34)16.

Dph3 of Saccharomyces cerevisiae interacts with Elp1-Elp3, although the Elongator complex is capable to form in the absence of Dph321. The Elongator complex interacts with RNA polymerase II and may play a role in transcription and transcriptional silencing4, 15, 22,23,24. However, studies in Schizosaccharomyces pombe have indicated that transcriptional defects of elp3Δ mutants are rather indirectly caused by inefficient translation of transcription factors such as Atf1 and Pcr125. Cells with defective Elongator lack mcm5U34 modifications at tRNALys UUU, tRNAGln UUG and tRNAGlu UUC, which results in inefficient translation of mRNAs with a high content of AAA, CAA, and GAA codons25,26,27. It has been shown that reduced protein levels in elp3Δ as well as sensitivity to H2O2, rapamycin and high temperature can be reversed by overexpression of tRNALys UUU 25, 26, 28. Impaired tRNA modifications in humans can lead to neurological diseases and various types of cancer29.

While there is evidence that lack of tRNA modifications cause a reduction in the levels of a subset of proteins, we know little about how simultaneous loss of Elp3-dependent tRNA modifications and of the Dph-dependent eEF2 diphthamide modification affects cell integrity. In the present study, we have addressed this by treating elp3Δ, dph3Δ and dph1Δ single and double mutants of S. pombe with drugs that induce DNA damage and different kinds of cytotoxic stress. Genetic analyses revealed that loss of tRNA modifications were responsible for most of the phenotypes. Our data suggest that Dph3 as part of the diphthamide synthesis pathway and in conjunction with Elp3-dependent tRNA modifications ensures efficient translation of key players of stress responses such as Atf1.

Results

dph3Δ and elp3Δ mutants were sensitive to a range of cytotoxic agents

We have previously shown that dph3Δ mutant cells were sensitive to hydroxyurea (HU), which depletes dNTP pools and thus stalls replication, and methyl methanesulfonate (MMS), which alkylates DNA and causes DNA breaks30. In this study, we were interested to know whether a defective dph3Δ renders cells sensitive to other cytotoxic agents or when exposed to thermal stress, and how other dphΔ mutants behave. We tested the dphΔ mutants as well as elp3Δ for sensitivity to HU, MMS, the microtubule depolymerising drug thiabendazole (TBZ), the TOR inhibitor rapamycin and for growth at low and high temperature. The dph3Δ, dph4Δ and dph1Δ mutants were sensitive to HU and MMS, while the other dphΔ mutants were not (Fig. 1A). The elp3Δ mutant was sensitive to MMS, TBZ, and rapamycin, showed reduced growth at 16 °C, and almost no growth at 37 °C. Among the dphΔ mutants, only dph3Δ was affected by rapamycin and cold (Fig. 1B). We further observed a subtle growth inhibition of dph3Δ in the presence of TBZ. While growth at 16 °C was rather delayed for dph3Δ and elp3Δ mutants, as they eventually caught up in growth with wild type, 37 °C appeared to kill most elp3Δ cells, since only a few grew to colonies when moved to 30 °C and incubated for another four days at this temperature (Supplementary Fig. S1).

Figure 1
figure 1

Drug and temperature sensitivity of dphΔ and elp3Δ mutants. (A) dph3Δ, dph4Δ and dph1Δ were sensitive to HU and MMS and elp3Δ was sensitive to MMS. Plates were incubated for three days at 30 °C. (B) The dph3Δ and elp3Δ mutants were sensitive to TBZ, rapamycin and low temperature. The elp3Δ mutant was extreme sensitive to 37 °C. Plates were incubated for two days at 30 °C, with the exceptions of one plate that was incubated for three days at 37 °C and one plate that was incubated for ten days at 16 °C. The control plate contained 0.04% DMSO.

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In our previous study, we have found that drug resistance markers integrated at the msh3 locus interfered with functions of the nearby located dph3 gene30. In contrast, cassettes deleting dph3 did not affect mating type switching, a process requiring Msh3. To ensure that drug and cold sensitivity of dph3Δ cells was not due to impaired Msh3 functions, caused by cassettes integrated at the dph3 locus, we tested a dph3-ATGmut strain for sensitivity to HU, MMS, TBZ, rapamycin and for growth at 16 °C. This strain contains a mutated ATG start codon without cassette integration30. We found that the dph3-ATGmut strain was, like dph3Δ, sensitive to all conditions tested (Supplementary Fig. S2). Thus, the observed phenotypes were due to a defective dph3.

Epistasis analysis of dph3Δ, dph1Δ and elp3Δ

Next, we performed epistasis analysis with dph3Δ, dph1Δ and elp3Δ. The dph3Δ single mutant was more sensitive to HU than dph1Δ and the elp3Δ single mutant behaved like wild type (Fig. 2A). The dph3Δ dph1Δ and dph3Δ elp3Δ double mutants and the dph3Δ dph1Δ elp3Δ triple mutant were similarly sensitive as the dph3Δ single mutant. This indicates that dph3Δ is epistatic with dph1Δ in response to HU. All three single mutants were sensitive to MMS, with dph3Δ being the most sensitive (Fig. 2A). The double mutants dph3Δ dph1Δ and dph3Δ elp3Δ and the triple mutant dph3Δ dph1Δ elp3Δ exhibited similar MMS sensitivity as the dph3Δ single mutant. The dph1Δ elp3Δ double mutant was slightly less sensitive, with a survival in the range of the dph1Δ and elp3Δ single mutants. Thus, dph3Δ, dph1Δ and elp3Δ were epistatic for MMS sensitivity. We further tested sensitivity to TBZ and rapamycin and growth at 16 °C and 37 °C. When treated with rapamycin or grown at 16 °C, dph3Δ and elp3Δ, but not dph1Δ, were affected (Fig. 2B). The dph3Δ elp3Δ double mutant showed a similar level of reduced growth at 16 °C as the dph3Δ and elp3Δ single mutants, indicating epistasis between these two deletions. Cells of dph3Δ and elp3Δ single, double and triple mutants, but not of the dph1Δ single mutant or of wild type, were elongated when grown at 16 °C (Fig. 3A). We further found that dph3Δ and dph1Δ rescued the growth defects of elp3Δ at 37 °C and in the presence of TBZ, and more subtly, sensitivity to rapamycin (Fig. 2B). The elp3Δ mutant grown at 37 °C had an increased number of cells with septa. 24% of the cells contained one septum, which was in some cells misplaced or relatively thick and 5% of the elp3Δ cells contained two or more septa (Table 1, Fig. 3B). Such a phenotype was not observed in wild type, dph1Δ or dph3Δ cells. When grown at 30 °C, the frequency of septated elp3Δ cells was as low as for wild type and the other mutant strains (Table 1). The nuclei of septated elp3Δ cells appeared to be like those of wild type cells without any abnormalities detectable, such as a “cut” (cell untimely torn) phenotype or unequal distribution of the genetic material (Fig. 3C). The occurrence of septated elp3Δ cells at 37 °C was reduced to the wild type level when dph3 and/or dph1 were additionally deleted (Table 1 and Fig. 3B). Our data indicate that the presence of Dph3 and Dph1 in the elp3Δ single mutant is detrimental to the cells when grown at 37 °C or exposed to TBZ.

Figure 2
figure 2

Epistasis and rescue effects with dph3Δ, dph1Δ and elp3Δ. (A) HU and MMS sensitivity. The dph3Δ dph1Δ and dph3Δ dph1Δ elp3Δ mutants showed similar sensitivity as the dph3Δ single mutant, indicating that Dph3 acts in the same pathways in response to HU and MMS as Dph1. The dph1Δ elp3Δ mutant was similarly sensitive to MMS as respective single mutants, indicating epistasis between dph1Δ and elp3Δ. The other double mutants and the triple mutant behaved like dph3Δ, indicating that dph3Δ is also epistatic with elp3Δ. Plates were incubated for three days at 30 °C. (B) dph3Δ and dph1Δ rescued the growth defect of elp3Δ at 37 °C and sensitivity to TBZ and rapamycin. Cold sensitivity at 16 °C appeared to be epistatic between dph3Δ and elp3Δ. The plate at 16 °C was incubated for ten days. The plate at 37 °C was incubated for three days. The other plates were incubated for two days. The control plate contained 0.033% DMSO.

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Figure 3
figure 3

Growth at 16 °C caused elongated dph3Δ and elp3Δ cells and growth at 37 °C caused septated elp3Δ cells, which was rescued by dph3Δ and dph1Δ. (A) dph3Δ and elp3Δ cells were elongated when grown at 16 °C. (B) elp3Δ cells frequently contained one or more septa when grown at 37 °C. This phenotype was not observed when dph3Δ and/or dph1Δ were additionally deleted. (C) Nuclei of septated elp3Δ cells were not untimely torn by septa. Shown are wild type and elp3Δ cells in phase contrast or stained with DAPI.

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Table 1 Increased number of septated elp3Δ cells at 37 °C.
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Rescue of drug and temperature sensitivity by elevated tRNALys UUU levels

It has been previously shown that an excess of unmodified tRNALys UUU encoded on a multi-copy plasmid suppressed sensitivity of elp3Δ to H2O2 and growth inhibition at 36 °C25, 26. We therefore tested whether elevated tRNALys UUU levels could complement the dph3Δ and elp3Δ phenotypes described in our study. We observed for dph3Δ that reduced growth at 16 °C and 37 °C and sensitivity to rapamycin was suppressed, whilst sensitivity to HU and TBZ remained largely unaffected (Fig. 4). A small effect was found in response to MMS. In the case of elp3Δ, elevated tRNALys UUU rescued all phenotypes, i.e. sensitivity to MMS, TBZ and rapamycin and growth inhibition at 16 °C and 37 °C. The dph3Δ elp3Δ double mutant containing the control plasmid pREP1 was less sensitive to TBZ than elp3Δ + pREP1, indicating rescue of elp3Δ sensitivity by dph3Δ, consistent with our previous findings (Fig. 2). dph3Δ elp3Δ + ptRNALys UUU was even more resistant to TBZ than dph3Δ elp3Δ + pREP1, and also more resistant than elp3Δ + ptRNALys UUU (Fig. 4B). Thus, tRNALys UUU complemented TBZ sensitivity of elp3Δ independently of dph3Δ. A rescue effect was also observed for the rapamycin treated double mutant, although here, the effects of dph3Δ and ptRNALys UUU may not be independently of each other.

Figure 4
figure 4

Rescue of dph3Δ and elp3Δ phenotypes by increased tRNALys UUU levels. Strains containing either the vector pREP1 or a plasmid expressing the gene for tRNALys UUU (SPBTRNALYS.06) were spotted on minimal medium with the indicated drugs and 5 μg/mL thiamine to repress the nmt promoter of pREP1. (A) HU and MMS sensitivity. (B) TBZ and rapamycin sensitivity and growth at 16 °C. The control plate contained 0.04% DMSO. (C) Growth at 37 °C. Excess of tRNALys UUU was able to rescue sensitivity of elp3Δ to MMS, TBZ, rapamycin and growth at 16 °C and 37 °C. In the case of dph3Δ, sensitivity to rapamycin and growth at 16 °C and 37 °C was rescued. Plates were incubated for three days, except the plate containing rapamycin and the plate at 16 °C, which were incubated for two and eleven days, respectively. (D) Increased tRNALys UUU levels lowered the number of septated elp3Δ cells at 37 °C. 5.5% of elp3Δ + ptRNALys UUU cells contained one septum, whereas 17.3% of elp3Δ + pREP1 cells had one septum (p = 0.002). 1.2% of elp3Δ + pREP1 cells and none of elp3Δ + ptRNALys UUU cells had two or more septa (p = 0.057; not significant). Columns represent averages of three independent counts of 100 or 200 cells with standard deviations.

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We noticed that at 37 °C dph3Δ grew significantly better on complex medium than on minimal medium and that the opposite was true for elp3Δ (Figs 1, 2 and 4). We observed an elevated number of septated cells of elp3Δ transformed with pREP1 (Fig. 4D). However, it was significantly lower than for elp3Δ grown on complex medium (Table 1) (18.5 ± 2.3% versus 29.5 ± 2.5%; p = 0.002). The presence of high tRNALys UUU levels in elp3Δ cells reduced the occurrence of septated cells to 5.5 ± 2.5% (p = 0.0027) (Fig. 4D). Thus, growth inhibition and septation at 37 °C either appeared to be dependent on the medium or was due to the presence of plasmids.

To test this and to demonstrate that the rescue effects were indeed due to the presence of ptRNALys UUU, we isolated elp3Δ mutants that had lost their plasmids (pREP1 or ptRNALys UUU) by culturing them in complex medium and isolating leu1-32 auxotrophic strains. Such strains were spotted on minimal medium supplemented with leucine and were grown at either 30 °C or 37 °C (Supplementary Fig. S3A). At 37 °C, all elp3Δ mutants showed growth inhibition and an increased number of septated cells when compared to wild type (Supplementary Table S1). We conclude that the presence of ptRNALys UUU in elp3Δ cells rescued temperature sensitivity and prevented accumulation of septated cells and that loss of the plasmid rendered the mutant sensitive again and caused a higher number of septated cells. In addition, we compared growth of dph3Δ and elp3Δ transformed with the vector pREP1 at 37 °C with untransformed mutant strains and did not found a relevant difference (Supplementary Fig. S3B).

The ribosomal rpl42-P56Q mutation caused MMS and HU sensitivity, which was suppressed by dph3Δ and elp3Δ

The rpl42-P56Q mutation of the 60 S ribosomal protein L42 interferes with translation elongation, likely through altered contacts with tRNAs31. We used this mutation to test genetic interaction with dph3Δ and elp3Δ. The rpl42-P56Q mutant itself was sensitive to HU and MMS, but not to TBZ, rapamycin or thermal stress (Fig. 5). MMS sensitivity of rpl42-P56Q was rescued by dph3Δ and elp3Δ. We further observed that elp3Δ complemented HU sensitivity of rpl42-P56Q. In the case of dph3Δ, we found either a weak suppression or epistasis with rpl42-P56Q, as the double mutant exhibited sensitivity like dph3Δ, and close to rpl42-P56Q (Fig. 5A). On the other hand, rpl42-P56Q rescued temperature sensitivity of elp3Δ. Since the dph3Δ elp3Δ rpl42-P56Q triple mutant grew better at 37 °C than the dph3Δ elp3Δ and elp3Δ rpl42-P56Q double mutants, suppression by rpl42-P56Q likely occurred independently of dph3Δ mediated rescue.

Figure 5
figure 5

Genetic interactions of dph3Δ and elp3Δ with rpl42-P56Q. (A) rpl42-P56Q was sensitive to HU and MMS. MMS sensitivity was rescued by dph3Δ and elp3Δ. HU sensitivity was rescued by elp3Δ. Here, the genetic relationship between dph3Δ and rpl42-P56Q is less clear. The mutations were either epistatic or dph3Δ showed a minor suppression effect. (B) The growth defect of elp3Δ at 37 °C was independently rescued by rpl42-P56Q and dph3Δ.

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Global protein levels were not significantly affected in elp3Δ and dph3Δ mutants

Translation of mRNAs is affected in elp3Δ and dph3Δ due to lack of tRNA modifications and in the latter mutant also due to lack of the diphthamide modification of eEF21,2,3,4,5, 16. We therefore tested global protein levels of dph3Δ and elp3Δ single and double mutants grown at 30 °C and 37 °C. We could not detect any significant differences to wild type at either temperature (Supplementary Fig. S4). Thus, under the conditions tested general protein synthesis appeared to be not affected in dph3Δ and elp3Δ mutants.

elp3Δ and dph3Δ showed positive genetic interactions with the stress response mutants sty1Δ and atf1Δ

The S. pombe transcription factors Atf1 and Pcr1 have been identified as translational targets of Elp325. Atf1 and Pcr1 protein levels were increased in H2O2 treated wild type cells, but remained low in an elp3Δ mutant. Atf1-Pcr1 is activated by the MAP kinase Sty1 in response to cellular stress32. Here, we tested sensitivity of sty1Δ, atf1Δ and pcr1Δ mutants to cytotoxic stress and performed epistasis analysis with elp3Δ and dph3Δ. The rationale was that if a double mutant was not more sensitive than the most sensitive single mutant, then the phenotypes of the elp3Δ and dph3Δ single mutants were (at least partially) due to low protein levels of the transcription factors Atf1 or Pcr1. In sty1Δ mutants, Atf1 cannot be activated by phosphorylation, display a low protein level, and thus, transcriptional activation of stress response genes by Atf1 is diminished32. We have found that sty1Δ and atf1Δ mutants were sensitive to HU, MMS, TBZ, and rapamycin and exhibited slow growth at 16 °C (Fig. 6A). MMS sensitivity and growth at 16 °C of the elp3Δ sty1Δ and elp3Δ atf1Δ double mutants was comparable to that of the single mutants, indicating epistasis of elp3Δ with sty1Δ and atf1Δ. In the cases of TBZ and rapamycin, the double mutants showed sensitivity similar to the sty1Δ and atf1Δ single mutants, which were less sensitive than elp3Δ. This difference could be due to detrimental effects of inefficient synthesis of Atf1 in the elp3Δ single mutant, which, for example, could lead to misfolded Atf1 protein, which interferes with cellular functions. For HU sensitivity, we did not obtain information about genetic interactions, since the elp3Δ single mutant was not sensitive.

Figure 6
figure 6

Genetic interactions of elp3Δ and dph3Δ with stress response mutations. (A) sty1Δ and atf1Δ showed positive genetic interactions (epistasis or weak rescue) with elp3Δ for MMS, TBZ, rapamycin and cold sensitivity. (B) dph3Δ was epistatic with atf1Δ for rapamycin and cold sensitivity. For TBZ a mild rescue was observed. The pcr1Δ mutant was cold sensitive independently of elp3Δ. (C) A dph3Δ atf1Δ double mutant was more sensitive to HU and MMS than respective single mutants, indicating independent functions of Dph3 and Atf1 in response to these drugs. Growth of pcr1Δ at 37 °C was inhibited, but rescued by dph3Δ. (D) The pcr1Δ and elp3Δ single mutants, but not the elp3Δ pcr1Δ and dph3Δ pcr1Δ double mutants, frequently had septated cells. Columns represent averages with standard deviations of at least three independent experiments, each with 200 counted cells. Values for wild type and dph3Δ were taken from Table 1. p-values for the occurrence of septated cells versus wild type were 2.2 × 10−7 for elp3Δ and 0.001 for pcr1Δ. p-values of elp3Δ pcr1Δ were 3.3 × 10−8 and 3.1 × 10−4 versus elp3Δ and pcr1Δ, respectively. The p-value of dph3Δ pcr1Δ versus pcr1Δ was 8.9 × 10−4.

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dph3Δ was epistatic with atf1Δ for rapamycin sensitivity and for growth inhibition at 16 °C (Fig. 6B), but not for HU or MMS, where the double mutant was clearly more sensitive than either single mutant (Fig. 6C). Thus, we found epistasis for MMS survival between elp3Δ and dph3Δ (Fig. 2A), between elp3Δ and atf1Δ (Fig. 6A), but not between dph3Δ and atf1Δ (Fig. 6C). The simplest explanation of the observed genetic interactions is that Dph3 and Atf1 likely have Elp3-independent functions in response to MMS, which are also independent to each other. This is consistent with the observations that the dph3Δ and atf1Δ single mutants were more sensitive to MMS than elp3Δ (Fig. 6C). For TBZ, we found a mild rescue effect of dph3Δ by atf1Δ (Fig. 6B).

The pcr1Δ single mutant was not sensitive to any of the cytotoxic agents tested (Fig. 6B,C), but growth was inhibited at 37 °C, which was rescued by additionally deleting dph3 (Fig. 6C). Like elp3Δ, the pcr1Δ single mutant, but not the elp3Δ pcr1Δ and dph3Δ pcr1Δ double mutants, had an increased number of septated cells (Fig. 6D). The pcr1Δ mutant also grew slowly at 16 °C (Fig. 6B). Since the elp3Δ pcr1Δ double mutant grew slower than either single mutant, cold sensitivity of elp3Δ was independent of pcr1Δ, and thus likely not due to low Pcr1 levels.

Taken together, survival of cells treated with HU, MMS, TBZ or rapamycin or when grown at 16 °C required the stress response factors Atf1 and Sty1. Since Atf1 has been shown to be regulated on the translational level by Elp3 via tRNA modifications25, and since the elp3Δ atf1Δ and the elp3Δ sty1Δ double mutants revealed epistasis, it is likely that phenotypes of elp3Δ were -to some extent- due to low Atf1 levels. This is also true for dph3Δ for TBZ, rapamycin and cold sensitivity.

Discussion

In the present study, we have analysed drug and thermal sensitivity caused by deletions of dph genes and elp3 in S. pombe. Dph proteins are required for diphthamide modification of eEF27. Elp3 is part of the Elongator complex, which is required for wobble uridine modifications of tRNAs2, 3, 15, 28. Dph3 participates in both types of modifications3, 7, 21 (Fig. 7). We have performed epistasis analysis and tested rescue effects mediated by elevated tRNALys UUU levels, by a ribosomal rpl42-P56Q mutation, and by dph3Δ and elp3Δ. In addition, genetic interactions of dph3Δ and elp3Δ with sty1Δ, atf1Δ and pcr1Δ have been analysed. The results are summarised in Tables 2 and 3.

Figure 7
figure 7

Model for the contribution of Dph3 in stress responses. Dph3 is supposed to be an electron donor for the Elp3-containing Elongator complex for modifications of wobble uridines in tRNA anticodons and for Dph1-Dph2 to initiate the first step of diphthamide modification of eEF2. Both modifications are required for efficient translation of mRNAs to proteins at ribosomes to produce key proteins involved in transcription, stress and DNA damage responses and other pathways that ensure accurate responses to cytotoxic agents and genome stability. One identified factor with reduced protein levels in elp3Δ background is Atf125. An atf1Δ mutant showed positive genetic interactions with elp3Δ and dph3Δ (Fig. 6).

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Table 2 Summary for drug and temperature sensitivity of dphΔ and elp3Δ mutants.
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Table 3 Summary of genetic interactions.
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Our study revealed that the dph1Δ, dph3Δ and dph4Δ mutants, but not dph2Δ, dph6Δ or dph7Δ, were sensitive to HU and MMS (Fig. 1 and Table 2). Thus, either Dph1 and Dph4 have additional functions apart from the eEF2 modification, or loss of the other Dph factors can be compensated. Interestingly, Dph1, Dph3 and Dph4 act in the first step of the diphthamide synthesis, whereas Dph6 and Dph7 are required for late steps1, 5,6,7. Thus, it could be that eEF2 containing intermediates of diphthamide is still functional with respect to accurate DNA damage responses, whereas unmodified eEF2 is not. Among the mutants tested, dph3Δ and elp3Δ showed the broadest phenotypic spectra (Table 2). These mutants were sensitive to low temperature, MMS, and possibly rapamycin in an epistatic relationship (Fig. 2 and Table 3). Such phenotypes were complemented by elevated tRNALys UUU levels (Fig. 4), indicating that lack of the mcm5U34 modification of this tRNA species caused sensitivity. However, in the case of MMS, also dph1Δ was sensitive, suggesting that this particular phenotype was either partially due to unmodified eEF2, or that Dph1 has a function in tRNA modifications. Indeed, we have found that dph1Δ and elp3Δ were epistatic for MMS sensitivity (Fig. 2), supporting the latter possibility.

The rpl42-P56Q mutation likely affects accurate and efficient interactions between tRNAs and ribosomes, a defect that is supposed to be also caused by unmodified tRNAs and eEF27, 27, 31, 33, 34. The rpl42-P56Q mutant was sensitive to MMS, which was partially suppressed by additional dph3Δ and elp3Δ deletions (Fig. 5). This may occur by slowing down translation due to lack of tRNA modifications, which compensates for impaired interactions between tRNAs and ribosomes caused by rpl42-P56Q. The elp3Δ mutation did not cause HU sensitivity, but allowed rescue of the HU sensitivity of rpl42-P56Q. dph3Δ was epistatic with dph1Δ for HU sensitivity, which was not suppressed by elevated tRNALys UUU levels (Table 3). Taken together, HU sensitivity is likely due to unmodified eEF2, which appears to affect translation in a similar way as the rpl42-P56Q mutation. Furthermore, we would like to point out that in certain cases the rpl42-P56Q mutation may not be suitable for genetic screens with S. pombe gene deletion libraries, since rpl42-P56Q itself is sensitive to MMS and HU and is able to rescue temperature sensitivity.

Although dph3Δ and elp3Δ shared most of their phenotypes, we found that elp3Δ was extreme sensitive to 37 °C and strongly affected by TBZ, whereas dph3Δ exhibited minor defects under such conditions. These were more pronounced on minimal medium, indicating a link between Dph3-dependent translational control and cellular metabolisms. It has been recently shown that lack of ncm5U34 and mcm5U34 tRNA modifications in elp3Δ mutants cause quantitative alterations in a wide range of metabolites35. It is therefore possible that different metabolic states in dependency of the growth media influenced the phenotypes of dph3Δ. Growth inhibition and septum formation of elp3Δ cells at 37 °C and TBZ sensitivity were suppressed by elevated tRNALys UUU levels, and also by dph3Δ and dph1Δ. We noticed that dph3Δ rescued growth of elp3Δ at 37 °C less than dph1Δ did (Fig. 2). This is likely not due to tRNA modification defects caused by dph3Δ, since the dph3Δ dph1Δ elp3Δ triple mutant showed full suppression. Increased tRNALys UUU levels and dph3Δ independently suppressed growth inhibition at 37 °C and TBZ sensitivity of elp3Δ (Fig. 4), indicating that the dph3Δ-dependent rescue effect was not due to unmodified tRNALys UUU. We conclude that lack of the eEF2 modification caused by dph1Δ and dph3Δ compensates for lack of tRNA modifications caused by elp3Δ.

A recent study revealed that S. pombe elp3Δ mutants were H2O2 sensitive and defective in uridine wobble modification of tRNALys UUU, tRNAGln UUG and tRNAGlu UUC, but were only mildly affected in transcription of H2O2–inducible genes25. The H2O2–inducible genes of the transcription factors Atf1 and Pcr1 contain a high number of lysine-encoding AAA versus AAG codons, when compared to highly expressed genes. Consequently, Atf1 and Pcr1 protein levels were low after H2O2 induced stress in an elp3Δ background, indicating inefficient translation of these proteins due to lack of tRNALys UUU wobble uridine modifications. In fact, overexpression of tRNALys UUU rescued H2O2 sensitivity of elp3Δ mutants25. Another study revealed that Elp3 of S. pombe is required for efficient translation of specific sets of proteins, including proteins involved in cell division control, as for example the kinase Cdr2, regulating the G2/M transition of the cell cycle26. Importantly, elevated tRNALys UUU levels rescued the growth defect of an elp3Δ ctu1Δ double mutant at 36 °C. Ctu1 is the homologue of S. cerevisiae Ncs6, which is involved in thiolation of wobble uridines in tRNAs for lysine, glutamine and glutamic acid; the same tRNAs that are modified by Elongator16. In our study, we found that among the mutants tested, elp3Δ showed the most prominent phenotypes, which were all rescued by elevated tRNALys UUU levels (Table 3). Elevated tRNALys UUU levels also suppressed dph3Δ phenotypes, with the exceptions of HU and TBZ sensitivity. Thus, the elp3Δ phenotypes and some of the dph3Δ phenotypes were at least to some extent caused by lack of the tRNALys UUU modifications. This likely resulted in inefficient biosynthesis of proteins required for stress responses as it has been reported for Cdr2, Atf1 and Pcr1 in an elp3Δ background25, 26. Our data support the previous studies in that an excess of unmodified tRNALys UUU can compensate for inefficient interactions with mRNAs. Importantly, we demonstrated that elp3Δ was epistatic with atf1Δ and sty1Δ for MMS and for growth inhibition at 16 °C, and showed positive genetic interactions (a mild rescue by atf1Δ and sty1Δ) for TBZ and rapamycin (Fig. 6). Thus, it is apparent that sensitivity of elp3Δ under these stress conditions was -at least partially- due to low levels of Atf1. As a consequence, transcriptional activation of stress response genes by the Atf1 transcription factor is affected. We further observed epistasis between dph3Δ and atf1Δ for rapamycin sensitivity and for growth inhibition at 16 °C and a mild rescue effect for TBZ, indicating that Dph3 ensures high Atf1 levels under such stress conditions. Although Atf1 acts as a heterodimer with Pcr132, we have found that atf1Δ was sensitive to all stress conditions we have tested, except for growth at 37 °C, whereas pcr1Δ was only inhibited for growth at low and high temperatures (Fig. 6). This is consistent with a previous study, which showed that atf1Δ, but not pcr1Δ, was sensitive to salt and osmotic stress, that Atf1 bound to some promoters in the absence of Pcr1, and that induction of some genes in response to high KCl concentrations required Atf1 but not Pcr136. Thus, the elp3Δ and dph3Δ phenotypes caused by low Atf1 levels, were likely due to loss of Pcr1-independent functions of Atf1 in regulating transcription of stress response genes.

Sensitivity of elp3Δ cells to high temperature may reflect an exacerbated situation, where not only lack of tRNA modification but also heat induced stress may affect protein biosynthesis, as it has been proposed to be the case for heat shock37, 38, or that protein homeostasis and/or folding is impaired at critical temperatures. It is known that in response to heat, the two subunits Ncs2 and Ncs6 of the Urm1 complex are downregulated through proteosomal degradation, which in turn affects thiolation of tRNAs and leads to altered protein levels39.

Defective tRNA modifications in humans have been associated with various diseases, including neurological and respiratory diseases, diabetes and different types of cancer29. For example, the human tRNA methyltransferase hTRM9L, which is required for methylating wobble uridines from cm5U34 to mcm5U34, is downregulated in various forms of carcinomas40. Re-expression in colon carcinoma cell lines allows suppression of tumour growth. Another human tRNA methyltransferase, hABH8 is required for resistance to the DNA damaging agents MMS and bleomycin41. Our data indicate that Elongator mediated tRNA wobble modifications and Dph catalysed diphthamide modification of eEF2 contribute to genome stability and stress responses in S. pombe. This is probably mediated by ensuring efficient translation of mRNAs to proteins that are required for the response to cytotoxic agents (Fig. 7). Some of the drugs we have tested here in relation to the status of elp3 and dph genes are frequently used in cancer chemotherapy. Mutations in DPH and ELP genes in humans may therefore serve as prognostic markers for the outcome of chemotherapy. In addition, drugs targeting DPH and ELP proteins may be developed. In combination with chemotherapeutic agents, this would allow improved growth inhibition of cancer cells.

Materials and Methods

Yeast media and genetic methods

Standard yeast genetic methods and media were as described elsewhere42. Yeast media were YEA (yeast extract agar), YEL (yeast extract liquid), MEA (malt extract agar), MMA (minimal medium agar), and EMM (Edinburgh minimal medium). YEA contained 0.5% yeast extract, 3% glucose, 1.6% agar. YEL had the same composition as YEA, but without agar. MEA contained 3% malt extract and 1.5% agar. MMA consisted of 0.17% yeast nitrogen base without amino acids/ammonium sulphate/thiamine, 0.5% ammonium sulphate, 1% glucose, and 1.8% granulated agar. EMM was prepared using 2.73% EMM broth without nitrogen, 0.5% ammonium chloride, and 2.5% granulated agar. The supplements adenine, histidine, uracil, and leucine were added at concentrations of 100 mg/L to complex media and MEA, and if required to MMA and EMM. 5 μg/mL thiamine was added to MMA and EMM if needed. G418 (100 mg/L), or hygromycin B (200 mg/L) were added to YEA after autoclaving and cooling down of the media where required. Yeast transformation was carried out by a modified protocol of Ito et al.43, omitting the use of potentially mutagenic carrier DNA. Crosses were performed by mixing h − with h + strains on the sporulation medium MEA, followed by incubation for 2–3 days at 30 °C. Suspensions of cells and spore-containing asci were subsequently treated for 30 min with 30% ethanol, which selectively kills vegetative cells, and then streaked on appropriate media. After growth, colonies of the progeny were transferred to master plates and genotypes of the strains were determined after replica plating on diagnostic media. Cells were analysed with an Axioskop 2 plus microscope (Zeiss) for taking images and otherwise with a Novex B microscope (euromex). The frequency of septated cells was calculated from at least three different counts (except for data shown in Supplementary Table S1), each including 100 or 200 cells.

Yeast strains

Yeast strains are listed in Supplementary Table S2. For strains used for spot tests or for counting septated cells, usually a second independently isolated strain with the same genotype (except possibly with the opposite mating type) was analysed. Some of those strains were not listed in Supplementary Table S2.

Strains SPAC8F11.02c (dph3Δ), SPAC21E11.03c (pcr1Δ), SPBC3B8.05 (dph1Δ), SPBC17D1.02 (dph2Δ), SPAC926.05 (dph4Δ), SPBC577.12 (dph6Δ), SPCC18.15 (dph7Δ), and SPAC29A4.20 (elp3Δ) were all from the Bioneer library44 version 2. The specified loci were disrupted by kanMX; the genetic background is h + ade6 (either allele M210 or M216) leu1-32 ura4-D18. An unrelated gene (SPAC26A3.16) has been previously named dph1 (http://www.pombase.org/)45. However, for simplification and consistency with the nomenclature for other organisms, we used here the gene name dph1 for locus SPBC3B8.05. Gene disruptions were confirmed by PCR. Also the sty1::ura4 gene disruption was verified by PCR using primers sty1-cp5 5′-TCTGGACAACAAAGCCTTGGTAAGA-3′, ura4_cPCR_For 5′-TTATAGATAAACACCTTGGG-3′ and ura4_R1 5′-TAAAGCAAGGGCATTAAGGC-3′ in a single reaction.

The elp3::hphMX strain DE33 was obtained by transformation of DE23 with a hphMX cassette containing PCR fragment amplified with primers Ptef-F 5′-CGCCAGCTGAAGCTTCGTAC-3′ and Ttef-R 5′-GGCCACTAGTGGATCTGATA-3′ using pFA6a-hphMX6 as template46.

Drug sensitivity tests

Drug sensitivities were analysed by spot tests as described previously30, except that we used here for most spot tests a pin multi replicator for spotting the different cell dilutions. Images were taken with a Gel Doc 2000 system (Bio-Rad) after two to three days of incubation at 30 °C, three days at 37 °C, or ten to eleven days at 16 °C. Drugs used in this study were hydroxyurea (Formedium), methyl methanesulfonate (ACROS Organics), thiabendazole (SIGMA) and rapamycin (LC Laboratories). 5 μg/mL thiamine was added to minimal medium used for spot tests with plasmid containing strains to suppress nmt promotor activity of pREP1.

DAPI staining

Cells were fixed overnight in 70% ethanol, spun down and washed with phosphate-buffered saline, spun down again and then dissolved in phosphate-buffered saline containing 4′,6-diamidino-2-phenylindole (1 μg/mL).

Cloning of SPBTRNALYS.06

The tRNALys UUU encoding gene SPBTRNALYS.06 together with 500 base pairs of upstream and downstream sequences was cloned into pREP147 following the procedure described by Fernández-Vázquez et al.25. In brief, the gene was amplified by PCR from genomic DNA with primers tRNALys-F 5′-ACTCTGCAGATGTCAAACAGTGGCAGATG-3′ and tRNALys-R 5′-TGAGAGCTCTTTCATACTTCTTTCGCCTC-3′, digested with PstI and SacI and ligated with pREP1, cut by the same restriction enzymes.

Plasmid loss of transformants

For loss of plasmids of transformed strains, 10 mL YEL was incubated with a loop-full of cell material and incubated on a shaker for two days at 30 °C. Aliquots were streaked on YEA and after growth, single colonies were tested for leucine auxotrophy.

Preparation and electrophoresis of protein extracts

S. pombe strains RO144 (wild type), DE4 (dph3Δ), DE23 (elp3Δ), and DE25 (dph3Δ elp3Δ) were grown in 20 mL YEL at 30 °C to a density of OD595nm of approximately 1. Aliquots of cells of the equivalent of 5 OD units were harvested by centrifugation, washed with 1 mL H2O and cell pellets were shock-frozen in liquid nitrogen and stored at −80 °C. The remaining cultures were shifted to 37 °C and aliquots were harvested after 30 min and after two hours, washed, frozen in liquid nitrogen and stored at −80 °C. 300 μL buffer H [25 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM β-mercaptoethanol, cOmplete EDTA-free protease inhibitor (Roche)] was added to the frozen cell pellets, which were then thawed on ice water. Cells were transferred to 2-mL screw cap tubes, mixed with 300 μL glass beads and were lysed by ten cycles of 20 sec at 6 meters/sec in a FastPrep-24 5 G (MP Biomedicals), with cooling on ice between each round. Samples were centrifuged for 15 min at 500 rpm at 4 °C to remove cell debris and intact cells. Lysates were centrifuged for 25 min at 14,000 rpm at 4 °C. Supernatants (soluble proteins) were kept on ice and pellets were suspended in buffer H, centrifuged for 20 min at 12,200 rpm at 4 °C and the resulting pellets (insoluble and chromatin bound proteins) suspended in buffer H in a volume equivalent to two thirds of the volume of the respective supernatant (approximately 120 μL). Aliquots of the fractions of soluble and insoluble proteins were boiled for 10 min at 96 °C in SDS-loading buffer and loaded on 14% SDS-polyacrylamide gels. After electrophoresis, gels were stained with InstantBlue (Expedeon). Gels were subsequently scanned and protein bands quantified with ImageJ software (NIH).