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. 2019 Aug 29;15(8):e1008117.
doi: 10.1371/journal.pgen.1008117. eCollection 2019 Aug.

SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications

Fu Xu  1 , Anders S Byström  1 , Marcus J O Johansson  1
Affiliations

SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications

Fu Xu et al. PLoS Genet. .

Abstract

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5/ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5/ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Increased expression of factors in the CWI signaling pathway counteracts the Ts phenotype of W303-derived elp3Δ cells.
(A) Growth of the elp3Δ (UMY3269) strain carrying the indicated high-copy (h.c.) or low-copy (l.c.) LEU2 plasmids. The high copy plasmid carrying the tK(UUU) and tQ(UUG) genes [74] is abbreviated as h.c. tK+tQ. Cells were grown over-night at 30°C in liquid synthetic complete medium lacking leucine (SC-leu), serially diluted, spotted on SC-leu plates, and incubated at 30°C or 37°C for 3 days. (B) Northern analysis of total RNA isolated from elp3Δ (UMY3269) cells carrying the indicated plasmids. The cells were grown in SC-leu medium at 30°C or 37°C. The blot was probed for tRNAUUULys, tRNAUUGGln, tRNAiMet, and 5.8S rRNA using radiolabeled oligonucleotides. 5.8S rRNA serves as the loading control.
Fig 2
Fig 2. The growth phenotypes elp3Δ cells are modulated by the allele at the SSD1 locus.
(A) Growth of elp3Δ mutants in the W303 and S288C genetic backgrounds. The wild-type (W303-1A and BY4741) and elp3Δ strains (UMY3269 and MJY1036) were streaked on SC plates and incubated at 30°C or 37°C for 3 days. (B) Effects of the ssd1-d2/SSD1 alleles on the growth of elp3Δ strains in the W303 and S288C genetic backgrounds. The ssd1-d2 (W303-1A and UMY4432), SSD1 (UMY3385 and BY4741), ssd1-d2 elp3Δ (UMY3269 and UMY4439) and SSD1 elp3Δ (UMY4456 and MJY1036) strains were grown over-night at 30°C in liquid SC medium, serially diluted, spotted on SC plates, and incubated at 30°C or 37°C for 3 days. (C) Effects of increased PKC1 dosage on the growth of elp3Δ ssd1-d2 and elp3Δ SSD1 strains. The relevant strains (UMY3269, UMY4456, UMY4439, and MJY1036) carrying the indicated plasmids were grown over-night at 30°C in liquid SC-leu medium, serially diluted, spotted on SC-leu plates, and incubated for 3 days at 30°C or 37°C. (D) Influence of the ssd1-d2 allele on growth phenotypes induced by various stress-inducing agents. The strains from B were grown over-night at 30°C in liquid SC medium, serially diluted, and spotted on SC plates and SC plates supplemented with caffeine, rapamycin, hydroxyurea or diamide. The plates were incubated for 3 days at 30°C.
Fig 3
Fig 3. The ssd1-d2 allele is required for the histone acetylation and telomeric gene silencing defects of elp3Δ cells.
(A) Western analysis of histones isolated from the ssd1-d2 (W303-1A), ssd1-d2 elp3Δ (UMY3269), SSD1 (UMY3385) and SSD1 elp3Δ (UMY4456) strains grown in SC medium at 30°C. Polyclonal anti-acetyl-histone H3 and anti-histone H3 antibodies were used to detect the indicated proteins. The blot is a representative of three independent experiments. (B) Influence of the ssd1-d2 allele on telomeric gene silencing in elp3Δ cells. The ssd1-d2 TELVIIL::URA3 (UMY2584) and ssd1-d2 elp3Δ TELVIIL::URA3 (UMY3790) strains carrying the indicated plasmids were grown over-night at 30°C in liquid SC-leu medium, serially diluted, spotted on SC-leu and SC-leu+5-FOA plates, and incubated for 3 days at 30°C.
Fig 4
Fig 4. The growth and protein homeostasis defects of elp3Δ ncs2Δ cells are augmented by the ssd1-d2 allele.
(A) Influence of the ssd1-d2 allele on the growth of elp3Δ ncs2Δ cells. The ssd1-d2 elp3Δ ncs2Δ (UMY4454 and MJY1159) and SSD1 elp3Δ ncs2Δ (UMY4467 and MJY1058) strains carrying the l.c. URA3 plasmid pRS316-ELP3 were grown over-night at 30°C in SC medium, serially diluted, spotted on SC and SC+5-FOA plates, and incubated for 3 days at 30°C. (B) Growth of S288C-derived SSD1 elp3Δ ncs2Δ and ssd1-d2 elp3Δ ncs2Δ strains. The wild-type (BY4741 and UMY4432) and elp3Δ ncs2Δ strains (MJY1058 and UMY4449) were streaked on a SC plate and incubated at 30°C for 2 days. (C) Effects of the ssd1-d2 allele on protein aggregation in elp3Δ ncs2Δ cells. Total protein and protein aggregates was analyzed from the ssd1-d2 (UMY4432), ssd1-d2 elp3Δ ncs2Δ (UMY4449), SSD1 (BY4741) and SSD1 elp3Δ ncs2Δ (MJY1058) strains grown in SC medium at 30°C. The gel is a representative of two independent experiments.
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Grants and funding

This work was supported by Magnus Bergvalls Foundation (2017-02098 to MJOJ); Åke Wibergs Foundation (M14-0207 to MJOJ); Swedish Research Council (621-2016-03949 to ASB); and Karin and Harald Silvanders Foundation/Insamlingsstiftelsen Umeå universitet (FS 2.1.6-1870-16 to ASB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.