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Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae.

Klassen R, Grunewald P, Thüring KL, Eichler C, Helm M, Schaffrath R - PLoS ONE (2015)

Bottom Line: Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect.Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU.These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.

ABSTRACT
In eukaryotes, wobble uridines in the anticodons of tRNA(Lys)UUU, tRNA(Glu)UUC and tRNA(Gln)UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

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tRNA overexpression suppression of phenotypes and protein depletion induced by lack of mcm5s2U.(A) Plasmid shuffling. The elp3urm1 strain carrying ELP3 on a centromeric URA3 plasmid was transformed with empty vector (pRS425) or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE) and subsequently streaked in parallel on –URA and FOA media. Above the FOA plate, sections corresponding to the indicated strains were magnified to illustrate colony sizes. (B) Serial dilution and spot assay with indicated strains. Replicas were incubated at 30°C or 37°C and photographed after 30h and again after 42h of incubation. (C) Serial dilution and spot assay with indicated strains on plates containing indicated amounts of rapamycin. Plates were incubated at 30°C and photographed after 44h. (D) Analysis of cellular protein content of WT and elp3uba4 strains transformed either with empty vecor (pRS425), or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE). Western detection of Pfk1 levels after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below.
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pone.0119261.g004: tRNA overexpression suppression of phenotypes and protein depletion induced by lack of mcm5s2U.(A) Plasmid shuffling. The elp3urm1 strain carrying ELP3 on a centromeric URA3 plasmid was transformed with empty vector (pRS425) or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE) and subsequently streaked in parallel on –URA and FOA media. Above the FOA plate, sections corresponding to the indicated strains were magnified to illustrate colony sizes. (B) Serial dilution and spot assay with indicated strains. Replicas were incubated at 30°C or 37°C and photographed after 30h and again after 42h of incubation. (C) Serial dilution and spot assay with indicated strains on plates containing indicated amounts of rapamycin. Plates were incubated at 30°C and photographed after 44h. (D) Analysis of cellular protein content of WT and elp3uba4 strains transformed either with empty vecor (pRS425), or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE). Western detection of Pfk1 levels after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below.

Mentions: First, we analyzed a potential suppression effect of excess levels of tRNALysUUU on the growth of an elp3urm1 double mutant immediately after counterselection of the ELP3-URA3 plasmid. The elp3urm1 [ELP3-URA3] strain was transformed with plasmids overexpressing tRNALysUUU alone (pK) or a combination of tRNAGlnUUG, tRNALysUUU and tRNAGluUUC (pQKE) as well as the empty vector (pRS425). All three strains grow equally well on SC-URA (maintaining the ELP3-URA3 plasmid) but exhibit very distinct growth phenotypes on FOA+URA, where the ELP3-URA3 plasmid cannot be maintained due to counter-selection. On the latter medium, the empty vector control shows small colonies. In contrast to this, both pK and pQKE carrying elp3urm1 strains exhibit significantly improved growth and colony sizes increased in the strain overexpressing the three tRNAs compared to the strain overexpressing tRNALys alone (Fig. 4A). Thus, higher-than-normal levels of tRNALysUUU lacking the wobble uridine modification have a significant suppressor effect on the slow growth of elp3urm1 cells, and this can be further enhanced by the additional overexpression of tRNAGlnUUG und tRNAGluUUC.


Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae.

Klassen R, Grunewald P, Thüring KL, Eichler C, Helm M, Schaffrath R - PLoS ONE (2015)

tRNA overexpression suppression of phenotypes and protein depletion induced by lack of mcm5s2U.(A) Plasmid shuffling. The elp3urm1 strain carrying ELP3 on a centromeric URA3 plasmid was transformed with empty vector (pRS425) or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE) and subsequently streaked in parallel on –URA and FOA media. Above the FOA plate, sections corresponding to the indicated strains were magnified to illustrate colony sizes. (B) Serial dilution and spot assay with indicated strains. Replicas were incubated at 30°C or 37°C and photographed after 30h and again after 42h of incubation. (C) Serial dilution and spot assay with indicated strains on plates containing indicated amounts of rapamycin. Plates were incubated at 30°C and photographed after 44h. (D) Analysis of cellular protein content of WT and elp3uba4 strains transformed either with empty vecor (pRS425), or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE). Western detection of Pfk1 levels after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below.
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pone.0119261.g004: tRNA overexpression suppression of phenotypes and protein depletion induced by lack of mcm5s2U.(A) Plasmid shuffling. The elp3urm1 strain carrying ELP3 on a centromeric URA3 plasmid was transformed with empty vector (pRS425) or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE) and subsequently streaked in parallel on –URA and FOA media. Above the FOA plate, sections corresponding to the indicated strains were magnified to illustrate colony sizes. (B) Serial dilution and spot assay with indicated strains. Replicas were incubated at 30°C or 37°C and photographed after 30h and again after 42h of incubation. (C) Serial dilution and spot assay with indicated strains on plates containing indicated amounts of rapamycin. Plates were incubated at 30°C and photographed after 44h. (D) Analysis of cellular protein content of WT and elp3uba4 strains transformed either with empty vecor (pRS425), or vectors overexpressing tRNALysUUU alone (pK) or together with tRNAGlnUUG and tRNAGluUUC (pQKE). Western detection of Pfk1 levels after chemical lysis of identical numbers of cells. Signal intensities relative to WT are indicated below.
Mentions: First, we analyzed a potential suppression effect of excess levels of tRNALysUUU on the growth of an elp3urm1 double mutant immediately after counterselection of the ELP3-URA3 plasmid. The elp3urm1 [ELP3-URA3] strain was transformed with plasmids overexpressing tRNALysUUU alone (pK) or a combination of tRNAGlnUUG, tRNALysUUU and tRNAGluUUC (pQKE) as well as the empty vector (pRS425). All three strains grow equally well on SC-URA (maintaining the ELP3-URA3 plasmid) but exhibit very distinct growth phenotypes on FOA+URA, where the ELP3-URA3 plasmid cannot be maintained due to counter-selection. On the latter medium, the empty vector control shows small colonies. In contrast to this, both pK and pQKE carrying elp3urm1 strains exhibit significantly improved growth and colony sizes increased in the strain overexpressing the three tRNAs compared to the strain overexpressing tRNALys alone (Fig. 4A). Thus, higher-than-normal levels of tRNALysUUU lacking the wobble uridine modification have a significant suppressor effect on the slow growth of elp3urm1 cells, and this can be further enhanced by the additional overexpression of tRNAGlnUUG und tRNAGluUUC.

Bottom Line: Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect.Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU.These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.

ABSTRACT
In eukaryotes, wobble uridines in the anticodons of tRNA(Lys)UUU, tRNA(Glu)UUC and tRNA(Gln)UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

Show MeSH
Related in: MedlinePlus