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Loss of ncm 5 and mcm 5 wobble uridine side chains results in an altered metabolic profile

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu, which in wild-type strains are modified with mcm5s2U, partially suppress phenotypes of an elp3Δ strain.

Objectives: To identify metabolic alterations in an elp3Δ strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu.

Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3Δ strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu. The temperature sensitive elp3Δ strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions.

Results: Culturing an elp3Δ strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3Δ strain carrying the wild-type ELP3 gene. Overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu suppressed a subset of the metabolic alterations observed in the elp3Δ strain.

Conclusion: Our results suggest that the presence of ncm5- and mcm5-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

Electronic supplementary material: The online version of this article (doi:10.1007/s11306-016-1120-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Overview of the pipeline for metabolic profiling of wild-type and elp3Δ strains carrying indicated plasmids. The UMY4238 and UMY4239 yeast strains contained either: an empty low-copy pRS315 vector (elp3Δ-l.c.-empty); a pRS315 vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3); an empty high copy pRS425 vector (elp3Δ-h.c.-empty, WT-h.c.-empty); or a high copy pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE, WT-h.c.-tKQE). Yeast strains were cultivated logarithmically to an OD600 value of ~0.5 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolite data was analyzed using multivariate analysis (PCA, PLS-DA) which separated the metabolites according to different classes representing the elp3Δ strains containing various plasmid constructs
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Fig1: Overview of the pipeline for metabolic profiling of wild-type and elp3Δ strains carrying indicated plasmids. The UMY4238 and UMY4239 yeast strains contained either: an empty low-copy pRS315 vector (elp3Δ-l.c.-empty); a pRS315 vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3); an empty high copy pRS425 vector (elp3Δ-h.c.-empty, WT-h.c.-empty); or a high copy pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE, WT-h.c.-tKQE). Yeast strains were cultivated logarithmically to an OD600 value of ~0.5 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolite data was analyzed using multivariate analysis (PCA, PLS-DA) which separated the metabolites according to different classes representing the elp3Δ strains containing various plasmid constructs

Mentions: We subjected an elp3Δ strain carrying either an empty low copy LEU2 vector (elp3Δ-l.c.-empty) or the same vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3) to metabolic profiling using non-targeted GC-TOF-MS (Fig. 1). This metabolic profiling was conducted to investigate whether loss of the ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in yeast tRNA causes metabolic alterations. We also included samples of the wild-type strain carrying an empty high-copy LEU2 vector (WT-h.c.-empty) to investigate whether metabolism of the elp3Δ-l.c.-ELP3 strain represents that of the wild-type strain.Fig. 1


Loss of ncm 5 and mcm 5 wobble uridine side chains results in an altered metabolic profile
Overview of the pipeline for metabolic profiling of wild-type and elp3Δ strains carrying indicated plasmids. The UMY4238 and UMY4239 yeast strains contained either: an empty low-copy pRS315 vector (elp3Δ-l.c.-empty); a pRS315 vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3); an empty high copy pRS425 vector (elp3Δ-h.c.-empty, WT-h.c.-empty); or a high copy pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE, WT-h.c.-tKQE). Yeast strains were cultivated logarithmically to an OD600 value of ~0.5 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolite data was analyzed using multivariate analysis (PCA, PLS-DA) which separated the metabolites according to different classes representing the elp3Δ strains containing various plasmid constructs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037161&req=5

Fig1: Overview of the pipeline for metabolic profiling of wild-type and elp3Δ strains carrying indicated plasmids. The UMY4238 and UMY4239 yeast strains contained either: an empty low-copy pRS315 vector (elp3Δ-l.c.-empty); a pRS315 vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3); an empty high copy pRS425 vector (elp3Δ-h.c.-empty, WT-h.c.-empty); or a high copy pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE, WT-h.c.-tKQE). Yeast strains were cultivated logarithmically to an OD600 value of ~0.5 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolite data was analyzed using multivariate analysis (PCA, PLS-DA) which separated the metabolites according to different classes representing the elp3Δ strains containing various plasmid constructs
Mentions: We subjected an elp3Δ strain carrying either an empty low copy LEU2 vector (elp3Δ-l.c.-empty) or the same vector containing the wild-type ELP3 gene (elp3Δ-l.c.-ELP3) to metabolic profiling using non-targeted GC-TOF-MS (Fig. 1). This metabolic profiling was conducted to investigate whether loss of the ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in yeast tRNA causes metabolic alterations. We also included samples of the wild-type strain carrying an empty high-copy LEU2 vector (WT-h.c.-empty) to investigate whether metabolism of the elp3Δ-l.c.-ELP3 strain represents that of the wild-type strain.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu, which in wild-type strains are modified with mcm5s2U, partially suppress phenotypes of an elp3Δ strain.

Objectives: To identify metabolic alterations in an elp3Δ strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu.

Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3Δ strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu. The temperature sensitive elp3Δ strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions.

Results: Culturing an elp3Δ strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3Δ strain carrying the wild-type ELP3 gene. Overexpression of hypomodified \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text {tRNA}_{{\rm s^{2} {\rm UUU}}}^{{\rm Lys}} , {\rm tRNA}_{{\rm s^{2} {\rm UUG}}}^{{\rm Gln }} \;{\rm and}\;{\rm tRNA}_{{\rm s^{2} {\rm UUC}}}^{{\rm Glu}}} $$\end{document}tRNAs2UUULys,tRNAs2UUGGlnandtRNAs2UUCGlu suppressed a subset of the metabolic alterations observed in the elp3Δ strain.

Conclusion: Our results suggest that the presence of ncm5- and mcm5-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

Electronic supplementary material: The online version of this article (doi:10.1007/s11306-016-1120-8) contains supplementary material, which is available to authorized users.

No MeSH data available.