Elp3 and Dph3 of Schizosaccharomyces pombe mediate cellular stress responses through tRNALys UUU modifications

Efficient protein synthesis in eukaryotes requires diphthamide modification of translation elongation factor eEF2 and wobble uridine modifications of tRNAs. In higher eukaryotes, these processes are important for preventing neurological and developmental defects and cancer. In this study, we used Schizosaccharomyces pombe as a model to analyse mutants defective in eEF2 modification (dph1Δ), in tRNA modifications (elp3Δ), or both (dph3Δ) for sensitivity to cytotoxic agents and thermal stress. The dph3Δ and elp3Δ mutants were sensitive to a range of drugs and had growth defects at low temperature. dph3Δ was epistatic with dph1Δ for sensitivity to hydroxyurea and methyl methanesulfonate, and with elp3Δ for methyl methanesulfonate and growth at 16 °C. The dph1Δ and dph3Δ deletions rescued growth defects of elp3Δ in response to thiabendazole and at 37 °C. Elevated tRNALys UUU levels suppressed the elp3Δ phenotypes and some of the dph3Δ phenotypes, indicating that lack of tRNALys UUU modifications were responsible. Furthermore, we found positive genetic interactions of elp3Δ and dph3Δ with sty1Δ and atf1Δ, indicating that Elp3/Dph3-dependent tRNA modifications are important for efficient biosynthesis of key factors required for accurate responses to cytotoxic stress conditions

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3- ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress

Composition of basic artificial wastewater (pH 7.0) used in the flow through reactor experiments.

<p>All components were the same during all experiments with the exception of Na₂SO₄ that was adjusted according the sulfate treatment used.</p

Relation of sulphide concetration and depth-integrated net sulphide production rates in wasterwater biofilms amended with different concentrations of nitrate (0–4 mM).

<p>(a) Mean H₂S concentration integrated in depth (μmol cm^-2) ± standard deviation and (b) depth-integrated net H₂S production rates at different concentrations of nitrate. Inserted graph presents the same data fitted to a linearized exponential decay equation (y = a e^{−b x}, ln y = ln a - bx), where b = -1.3840, ln a = -2.08, R² = 0.65 (at 2 mM sulfate) and b = -1.3869, ln a = -0.64 and R² = 0.75 (at 10 mM sulfate) (p values < 0.0001).</p

Vertical H₂S, O₂ and pH profiles in wastewater biofilm and modeled reaction rates.

<p>(a) Representative H₂S (triangles), O₂ (circles) and pH (diamonds) profiles in a biofilm at 1.5 mM sulfate and no nitrate; (b) and (c) modeled concentration profile (thick black lines) and volumetric reaction rates (grey straight lines) for O₂ and H₂S, respectively. Depths with the same rates determine the biofilm microzonation. Areal rates of O₂ consumption and net sulfide production are also indicated (nmol cm^-2 h^-1).</p

Vertical microprofiles of H₂S, O₂ and pH in wastewater biofilms amended with sulfate and nitrate.

<p>(A) Representative H₂S (triangles, O₂ (circles) and pH (diamonds) profiles in a biofilm with 10 mM sulfate and 1 mM nitrate and modeled profiles of (B) O₂ and (C) H₂S. Real data represented with symbols and modeled profiles with bold black lines. Boxes (grey lines) represent volumetric respiration and sulfide production profiles (μmol cm^-3 h^-1). Depths with the same rates determine the biofilm microzonation. (B) Areal rates of O₂ consumption and (C) net sulfide production (nmol cm^-2 h^-1). (D, E and F) Changes in H₂S concentration, modeled profiles and volumetric net sulfide production rates with depth are shown in lower panels at 10 mM sulfate and increasing nitrate concentrations from 0 to 1 mM nitrate.</p

Net mass balance production rates of nitrite and nitrate in wastewater biofilms amended with varying concetrations of sulfate and nitrate.

<p>(a) Relation between the nitrite production by the biofilm and increasing sulfate concentration in the bulk water and no added nitrate and (b) on increasing nitrate concentrations in the presence of 2 and 10 mM sulfate. (c) Relation between nitrate production by the biofilm and increasing nitrate concentrations in the presence of 2 and 10 mM sulfate. Inserted in the plots are the slopes of the regression lines (a), the intercepts (b) and determination coefficients (R²).</p

Sulfide dynamics in wastewater biofilms.

<p>(a) Representative vertical H₂S concentration profiles with increasing sulfate concentrations. (b) Example of evolution of sulfide concentration profiles over time in a biofilm subjected to an increase of sulfate concentration from 0.5 to 1.5 mM. (c) Net areal sulfide production (first 1.5 mm of the biofilm) with increasing sulfate concentrations. (d) Mean H₂S concentration integrated in depth (μmol cm^-2) ± standard deviation with increasing concentrations of sulfate (n = 1 for 0.5 and 20 mM SO₄^2-, n = 4 for 6 and 15 mM, n = 5 for the rest). Integrated mean H₂S concentrations were calculated from measured H₂S profile from the surface up to a depth of 1.5 mm within the biofilm.</p

Microbial community DGGE profiles for cDNA extracted from waster water biofilms before and after addition of varying concentrations of sulfate and nitrate.

<p>Samples are from biofilms incubated with 2 mM sulfate and increasing amounts of nitrate (2 + ΔN), 10 mM sulfate and increasing amount of nitrate (10 + ΔN), and increasing amount of sulfate and no addition of nitrate to the artificial wastewater (ΔS). Samples were collected before (bef) and 12 hours after (aft) the beginning of nitrate or sulfate addition. Column M represents the marker.</p

Maximum oxygen penetration depth in wastewater biofilms.

<p>(a) Maximum oxygen penetration depth (Zox) in the biofilm under different sulfate concentrations in the bulk water phase and (b) Maximum oxygen penetration depth (Zox) in the biofilm as a function of sulfide production rate in the upper production layer. Bars represent standard deviation of means (O2 penetration depth from n = 2 (penetration values at 0.5 and 20 mM), 8 (values at 6 and 15 mM) or 10 (the rest) profiles.). Inserted in every plot is the slope of the regression line (a), the intercept (b) and determination coefficient (R2).</p