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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.


Related in: MedlinePlus

Metabolic variation of the wild-type and elp3Δ strain containing indicated plasmids. The UMY4238 and UMY4239 yeast strains containing 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) were grown logarithmically to an OD600 of ~0.5 at 30 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Each dot in the PCA analysis represents a technical replicate from three different biological replicates
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Fig2: Metabolic variation of the wild-type and elp3Δ strain containing indicated plasmids. The UMY4238 and UMY4239 yeast strains containing 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) were grown logarithmically to an OD600 of ~0.5 at 30 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Each dot in the PCA analysis represents a technical replicate from three different biological replicates

Mentions: Since elp3Δ strains are temperature sensitive (Ts), we cultured strains to ~0.5 OD600 units under permissive (30 °C) and semi-permissive (34 °C) growth conditions, as metabolic changes may be more pronounced at elevated temperatures. From our metabolite extracts, 111 metabolites could be measured using GC-TOF-MS, 41 of which could be identified while the remaining were unidentified. We performed a PCA of all the strains used and all 111 metabolites detected in this study to get an overview of metabolism in the strains. The PCA results showed that the elp3Δ-l.c.-ELP3 strain clusters with the WT-h.c.-empty strain, indicating that metabolism in these strains is similar (Fig. 2).Fig. 2


Loss of ncm 5 and mcm 5 wobble uridine side chains results in an altered metabolic profile
Metabolic variation of the wild-type and elp3Δ strain containing indicated plasmids. The UMY4238 and UMY4239 yeast strains containing 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) were grown logarithmically to an OD600 of ~0.5 at 30 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Each dot in the PCA analysis represents a technical replicate from three different biological replicates
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037161&req=5

Fig2: Metabolic variation of the wild-type and elp3Δ strain containing indicated plasmids. The UMY4238 and UMY4239 yeast strains containing 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) were grown logarithmically to an OD600 of ~0.5 at 30 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Each dot in the PCA analysis represents a technical replicate from three different biological replicates
Mentions: Since elp3Δ strains are temperature sensitive (Ts), we cultured strains to ~0.5 OD600 units under permissive (30 °C) and semi-permissive (34 °C) growth conditions, as metabolic changes may be more pronounced at elevated temperatures. From our metabolite extracts, 111 metabolites could be measured using GC-TOF-MS, 41 of which could be identified while the remaining were unidentified. We performed a PCA of all the strains used and all 111 metabolites detected in this study to get an overview of metabolism in the strains. The PCA results showed that the elp3Δ-l.c.-ELP3 strain clusters with the WT-h.c.-empty strain, indicating that metabolism in these strains is similar (Fig. 2).Fig. 2

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.


Related in: MedlinePlus