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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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Deletion of the tRNAGlnCUG gene (ΔtQ(CUG)) causes inviability in WT and elp3uba4 double mutants.WT and elp3uba4 strains were transformed with the single copy URA3 plasmid pAK01 carrying the tRNAGlnCUG gene and subsequently, the genomic copy of the gene was deleted. Strains were grown in parallel on –URA and FOA medium.
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pone.0119261.g002: Deletion of the tRNAGlnCUG gene (ΔtQ(CUG)) causes inviability in WT and elp3uba4 double mutants.WT and elp3uba4 strains were transformed with the single copy URA3 plasmid pAK01 carrying the tRNAGlnCUG gene and subsequently, the genomic copy of the gene was deleted. Strains were grown in parallel on –URA and FOA medium.

Mentions: We first checked morphology of the elp3uba4 double mutant in comparison to the wild type (S1 Fig.). We found a small number of irregularly shaped cells containing elongated and/or multiple buds which were not observed for the isogenic wild type. There were no other significant differences between wild type and the elp3uba4 double mutant with respect to cell size or nuclear morphology (S1 Fig.). As an initial test to the functional role of mcm5s2U, we asked whether the complete absence of the modification might affect the ability of tRNAGlnUUG, to recognize non-cognate codons. It was shown previously that mcm5s2U modified tRNAGlnUUG is able to decode the alternative CAG codon via U/G wobbling when present at elevated levels but not under normal circumstances [3]. Thus, high copy tRNAGlnUUG suppresses the lethal effect of a deletion in SUP70, the only gene for tRNAGlnCUG [3]. Since tRNALeuUAG, the only known yeast tRNA naturally carrying an unmodified wobble uridine is capable of efficient decoding of all four CUN codons by wobble base pairing [2, 31], it appeared possible that complete absence of wobble uridine modifications might also increase the ability of tRNAGlnUUG to decode the CAG codon via U/G wobbling. To test this idea, we introduced a URA3 plasmid carrying the tRNAGlnCUG gene into WT and elp3uba4 double mutants and subsequently deleted the single chromosomal gene for tRNAGlnCUG. On FOA medium, the URA3 plasmid-bourne tRNAGlnCUG cannot be maintained, resulting in complete growth arrest of WT cells due to the inability of tRNAGlnUUG to efficiently decode the CAG codon [3]. Growth assays further revealed that the elp3uba4 double deletion did not suppress the inviability on FOA medium (Fig. 2), indicating that tRNAGlnUUG carrying an unmodified wobble uridine is also inable to efficiently decode the CAG codon. Hence, mcm5s2U is not responsible for the general inability of tRNAGlnUUG to efficiently read the alternative G-ending codon.


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)

Deletion of the tRNAGlnCUG gene (ΔtQ(CUG)) causes inviability in WT and elp3uba4 double mutants.WT and elp3uba4 strains were transformed with the single copy URA3 plasmid pAK01 carrying the tRNAGlnCUG gene and subsequently, the genomic copy of the gene was deleted. Strains were grown in parallel on –URA and FOA medium.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352028&req=5

pone.0119261.g002: Deletion of the tRNAGlnCUG gene (ΔtQ(CUG)) causes inviability in WT and elp3uba4 double mutants.WT and elp3uba4 strains were transformed with the single copy URA3 plasmid pAK01 carrying the tRNAGlnCUG gene and subsequently, the genomic copy of the gene was deleted. Strains were grown in parallel on –URA and FOA medium.
Mentions: We first checked morphology of the elp3uba4 double mutant in comparison to the wild type (S1 Fig.). We found a small number of irregularly shaped cells containing elongated and/or multiple buds which were not observed for the isogenic wild type. There were no other significant differences between wild type and the elp3uba4 double mutant with respect to cell size or nuclear morphology (S1 Fig.). As an initial test to the functional role of mcm5s2U, we asked whether the complete absence of the modification might affect the ability of tRNAGlnUUG, to recognize non-cognate codons. It was shown previously that mcm5s2U modified tRNAGlnUUG is able to decode the alternative CAG codon via U/G wobbling when present at elevated levels but not under normal circumstances [3]. Thus, high copy tRNAGlnUUG suppresses the lethal effect of a deletion in SUP70, the only gene for tRNAGlnCUG [3]. Since tRNALeuUAG, the only known yeast tRNA naturally carrying an unmodified wobble uridine is capable of efficient decoding of all four CUN codons by wobble base pairing [2, 31], it appeared possible that complete absence of wobble uridine modifications might also increase the ability of tRNAGlnUUG to decode the CAG codon via U/G wobbling. To test this idea, we introduced a URA3 plasmid carrying the tRNAGlnCUG gene into WT and elp3uba4 double mutants and subsequently deleted the single chromosomal gene for tRNAGlnCUG. On FOA medium, the URA3 plasmid-bourne tRNAGlnCUG cannot be maintained, resulting in complete growth arrest of WT cells due to the inability of tRNAGlnUUG to efficiently decode the CAG codon [3]. Growth assays further revealed that the elp3uba4 double deletion did not suppress the inviability on FOA medium (Fig. 2), indicating that tRNAGlnUUG carrying an unmodified wobble uridine is also inable to efficiently decode the CAG codon. Hence, mcm5s2U is not responsible for the general inability of tRNAGlnUUG to efficiently read the alternative G-ending codon.

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