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. 2013;9(7):e1003647.
doi: 10.1371/journal.pgen.1003647. Epub 2013 Jul 18.

Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs

Jorge Fernández-Vázquez  1 , Itzel Vargas-Pérez, Miriam Sansó, Karin Buhne, Mercè Carmona, Esther Paulo, Damien Hermand, Miguel Rodríguez-Gabriel, José Ayté, Sebastian Leidel, Elena Hidalgo
Affiliations

Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs

Jorge Fernández-Vázquez et al. PLoS Genet. 2013.

Abstract

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNA(Lys) UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cells lacking Sin3/Elp3 are sensitive to H2O2, but do not display major transcriptional defects.
(A) Δsin3/elp3 strain and other mutants of Elongator complex are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), AV18 (Δsty1), MS161 (Δgcn5), IV16 (Δsin3/elp3), MS98 (Δatf1), IV72 (Δelp4), IV66 (Δiki3/elp1) and IV68 (ΔSPAC30.02c; S. pombe ortholog of kti12) were spotted onto rich plates without (Untreated) or with the indicated concentrations of H2O2 or KCl, and grown at 30°C unless indicated (37°C). (B) Total levels of histone H3 acetylation at lysines 9 and 14 are not affected in a Δsin3/elp3 strain. Protein extracts from strains 972 (WT), MS161 (Δgcn5) and IV16 (Δsin3/elp3) were analyzed by Western blot with antibodies against acetylated Lys9 and Lys14 of histone H3 (H3K9K14-Ac) or total H3, as a loading control. (C) Deletion of clr3 does not rescue Δsin3/elp3 sensitivity to oxidative stress. Serial dilutions from cultures of strains 972 (WT), IV16 (Δsin3/elp3), SPK19 (Δclr3) and IV95 (Δsin3/elp3 Δclr3) were spotted onto rich plates without (Untreated) or with 2 mM H2O2. (D) Stress-dependent transcriptional analysis of wild-type and Δsin3/elp3 cells. Culture of strains 972 (WT), MS161 (Δgcn5) and IV16 (Δsin3/elp3) were treated with 1 mM H2O2 for the indicated times. Total RNA was analyzed by Northern blot with probes for ctt1, gpd1, hsp9, srx1, and gpx1. act1 is shown as a loading control. (E) Stress-dependent H3 acetylation at CESR genes does not require Sin3/Elp3. Cultures of strains 972 (WT) and IV16 (Δsin3/elp3) were treated (+) or not (−) with 1 mM H2O2 for 5 min. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies specific for acetylated Lys9 and Lys14 of histone H3 (H3Ac) or against unmodified C-terminal domain of H3 (H3). The percentage of immunoprecipitation of acetylated H3 versus total H3 is indicated (% IP H3Ac/H3). ChIP experiments were performed using primers covering promoter (prom), coding (ORF) and termination (term) sequences of the gpd1 gene. (F) Stress-dependent nucleosome eviction at CESR genes does not require Sin3/Elp3. The same experiment as in E is represented here as the percentage of immunoprecipitation of total H3 (%IP total H3). Error bars (SEM) for all ChIP experiments were calculated from biological triplicates.
Figure 2
Figure 2. Sin3/Elp3 is required for modification of uridine-34 (U34) at the anticodon of some cytoplasmic tRNAs.
(A) Sin3/Elp3 and the Ctu1-Ctu2 pathways are required for the mcm5 and s2 modifications, respectively, at U34 of some anticodons. (B) Northern blot analysis of bulk tRNA isolated from WT (972), IV16 (Δsin3/elp3), YDH 644 (Δctu1), IV86 (Δctu2), and YDH 254 (Δctu1 Δctu2) using specific probes against tRNALys UUU, tRNAGln UUG, tRNAGlu UUC and tRNAArg UCU (negative control) by the APM-gel retardation method. The position of the unmodified (tRNA) or modified (mcm5s2 tRNA) tRNAs are indicated with arrows. (C) Δctu1 and Δctu2 strains are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), AV18 (Δsty1), IV16 (Δsin3/elp3), IV86 (Δctu2), YDH 644 (Δctu1), YDH 254 (Δctu1 Δctu2) were spotted onto rich plates without (Untreated) or with 1 mM H2O2. (D) Double mutant Δsin3/elp3 Δctu2 colonies are similar in size to single mutant Δsin3/elp3. Tetrad analysis of a cross between IV16 (Δsin3/elp3) and IV86 (Δctu2) strains. Each vertical box corresponds to the four spores of a tetrad. The genotypes of each colony of the top panel are indicated in the corresponding positions of the bottom panel: WT, Δctu2 (Δc), Δsin3/elp3 (Δe) or Δsin3/elp3 Δctu2 (ΔΔ). (E) Single mutant Δsin3/elp3 and double mutant Δsin3/elp3 Δctu2 show similar sensitivity to oxidative stress. Serial dilutions from cultures of strains 972 (WT), IV16 (Δsin3/elp3) and JF73 (Δsin3/elp3 Δctu2) were spotted onto rich plates without (Untreated) or with 1 mM H2O2.
Figure 3
Figure 3. Over-expression of tRNALys UUU supresses the growth defects of Δsin3/elp3 upon oxidative stress.
(A) Relative levels of tRNA over-expression by Northern blot. Total RNA from strains 972 (WT), IV16 (Δsin3/elp3), or JF77 (Δsin3/elp3) transformed with episomal plasmids p465 (ptRNALys UUU), p466 (ptRNALys CUU), p467 (ptRNAGln UUG), p468 (ptRNAGlu UUC) or the empty vector pREP.42x, was analyzed by Northern blot with probes of dsDNA of the indicated tRNAs labeled at their antisense strand. act1 is shown as a loading control. The numbers below last panel indicate the relative levels of the corresponding over-expressed tRNA normalized to basal levels in wild-type strain. (B) Serial dilutions from cultures of strains as in A were spotted onto synthetic rich media plates without (Untreated) or with 2 mM H2O2.
Figure 4
Figure 4. Protein levels of the stress transcription factors Atf1 and Pcr1 depend on the U34 modifying activities Sin3/Elp3 and Ctu2.
(A) Scheme illustrating the activation of the stress gene expression program by Atf1 and Pcr1 (see text for details). (B and C) Absence of Sin3/Elp3 or Ctu2 barely affects transcription of the atf1 and pcr1 genes but largely affects Atf1 and Pcr1 protein levels. Rich media cultures of strains 972 (WT), IV16 (Δsin3/elp3) and IV86 (Δctu2) treated with 1 mM H2O2 at the indicated time points were analyzed to determine transcription levels of atf1 and pcr1 genes by Northern blot (B), or Atf1 and Pcr1 protein levels by Western blot using polyclonal antibodies (C). act1 mRNA or tubulin were used as loading controls for B and C, respectively. (D) Quantification of the relative mRNA and protein levels for atf1, pcr1, Atf1 and Pcr1 in wild-type and mutant strains. The Northern or Western blot panels of experiments as in B and C, respectively, were quantified and represented here relative to untreated wild-type levels (with an assigned value of 1). The atf1 and pcr1 mRNA levels normalized to act1 are shown in the left two panels, whereas the Atf1 and Pcr1 protein levels normalized to tubulin are shown in the two right panels. Error bars (SEM) were calculated from biological duplicates.
Figure 5
Figure 5. Expression of a synthetic AAA-to-AAG atf1 gene rendered wild-type Atf1 protein levels in Elongator mutants.
(A and B) Vectors carrying a constitutively expressed wild-type (pHA-atf1′) or mutated atf1 gene (pHA-atf1 AAG′) were integrated in the chromosomes of wild-type or Δsin3/elp3 strains. Rich media cultures of strains JF91 (WT+pHA-atf1′), JF92 (Δsin3/elp3+pHA-atf1′), JF94 (WT+pHA-atf1 AAG′) and JF95 (Δsin3/elp3+pHA-atf1 AAG′), either untreated (0) or treated with 1 mM H2O2 for the indicated times, were analyzed to determine HA-atf1 mRNA levels by Northern blot using an anti-HA probe (A) or HA-Atf1 protein levels by Western blot using monoclonal antibody against HA (B). The numbers below the Northern or Western blot panels indicate the relative levels of HA-atf1/act1 mRNAs (panel A) or HA-Atf1/tubulin protein levels (panel B). (C) Expression of the mutant AAA-to-AAG Atf1 protein does not suppress the growth defects of Δsin3/elp3 cells upon oxidative stress. Empty vector or plasmids carrying a wild-type (pHA-atf1′) or a mutated atf1 gene (pHA-atf1 AAG′) were integrated in the chromosomes of wild-type or Δsin3/elp3 strains. Cultures from the resulting strains JF88 (WT+empty vector), JF89 (Δsin3/elp3+empty vector), JF91 (WT+pHA-atf1′), JF92 (Δsin3/elp3+pHA-atf1′), JF94 (WT+pHA-atf1 AAG′) and JF95 (Δsin3/elp3+pHA-atf1 AAG′) were serially diluted and spotted onto rich media plates without (Untreated) or with 1 mM H2O2.
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