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

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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.


Metabolic alterations upon loss of ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in tRNA. The UMY4239 elp3Δ strains contained either: an empty pRS315 low copy vector (l.c. empty); a pRS315 vector carrying the wild-type ELP3 gene (l.c. ELP3); an empty pRS425 high copy vector (h.c. empty); or a pRS425 high copy vector carrying the tRNA genes tK(UUU); tQ(UUG) and tE(UUC) (h.c. tKQE). These yeast strains were grown logarithmically to ~0.5 OD600 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolites were hierarchically organized into five clusters as represented by a–e based on metabolite abundance (see Online Resource 2). Red signifies metabolite enrichment and green signifies metabolite reduction
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Fig5: Metabolic alterations upon loss of ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in tRNA. The UMY4239 elp3Δ strains contained either: an empty pRS315 low copy vector (l.c. empty); a pRS315 vector carrying the wild-type ELP3 gene (l.c. ELP3); an empty pRS425 high copy vector (h.c. empty); or a pRS425 high copy vector carrying the tRNA genes tK(UUU); tQ(UUG) and tE(UUC) (h.c. tKQE). These yeast strains were grown logarithmically to ~0.5 OD600 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolites were hierarchically organized into five clusters as represented by a–e based on metabolite abundance (see Online Resource 2). Red signifies metabolite enrichment and green signifies metabolite reduction

Mentions: A comparison of the metabolic profiles of only identified metabolites showed that permissive growth (30 °C) of the elp3Δ-l.c.-empty strain resulted in elevated levels of primarily Ornithine, Lysine and N-Acetylglucosamine, and reduced levels of Glutamine, Beta-alanine, Malic acid, Aspartic acid, Pyroglutamic acid, Alanine, Threonine, 2-Aminobutyric acid, 5,6-Dihydrouracil and Tyrosine when compared to the elp3Δ-l.c.-ELP3 strain (Fig. 5a–e, Online Resource 2–3). Semi-permissive growth (34 °C) of the elp3Δ-l.c.-empty strain also resulted in elevated levels of Ornithine, Lysine and N-Acetylglucosamine when compared to the elp3Δ-l.c.-ELP3 strain (Fig. 5a–e, Online Resource 2–3). We also observed that the elp3Δ-l.c.-empty strain accumulated eight additional metabolites with known identity at 34 °C (Online Resource 4), indicating more pronounced metabolic changes with growth at elevated temperatures.Fig. 5


Loss of ncm 5 and mcm 5 wobble uridine side chains results in an altered metabolic profile
Metabolic alterations upon loss of ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in tRNA. The UMY4239 elp3Δ strains contained either: an empty pRS315 low copy vector (l.c. empty); a pRS315 vector carrying the wild-type ELP3 gene (l.c. ELP3); an empty pRS425 high copy vector (h.c. empty); or a pRS425 high copy vector carrying the tRNA genes tK(UUU); tQ(UUG) and tE(UUC) (h.c. tKQE). These yeast strains were grown logarithmically to ~0.5 OD600 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolites were hierarchically organized into five clusters as represented by a–e based on metabolite abundance (see Online Resource 2). Red signifies metabolite enrichment and green signifies metabolite reduction
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Fig5: Metabolic alterations upon loss of ncm5U, mcm5U and mcm5s2U wobble uridine nucleosides in tRNA. The UMY4239 elp3Δ strains contained either: an empty pRS315 low copy vector (l.c. empty); a pRS315 vector carrying the wild-type ELP3 gene (l.c. ELP3); an empty pRS425 high copy vector (h.c. empty); or a pRS425 high copy vector carrying the tRNA genes tK(UUU); tQ(UUG) and tE(UUC) (h.c. tKQE). These yeast strains were grown logarithmically to ~0.5 OD600 at 30 or 34 °C and harvested. Metabolites were extracted and then quantified using GC-TOF-MS. Metabolites were hierarchically organized into five clusters as represented by a–e based on metabolite abundance (see Online Resource 2). Red signifies metabolite enrichment and green signifies metabolite reduction
Mentions: A comparison of the metabolic profiles of only identified metabolites showed that permissive growth (30 °C) of the elp3Δ-l.c.-empty strain resulted in elevated levels of primarily Ornithine, Lysine and N-Acetylglucosamine, and reduced levels of Glutamine, Beta-alanine, Malic acid, Aspartic acid, Pyroglutamic acid, Alanine, Threonine, 2-Aminobutyric acid, 5,6-Dihydrouracil and Tyrosine when compared to the elp3Δ-l.c.-ELP3 strain (Fig. 5a–e, Online Resource 2–3). Semi-permissive growth (34 °C) of the elp3Δ-l.c.-empty strain also resulted in elevated levels of Ornithine, Lysine and N-Acetylglucosamine when compared to the elp3Δ-l.c.-ELP3 strain (Fig. 5a–e, Online Resource 2–3). We also observed that the elp3Δ-l.c.-empty strain accumulated eight additional metabolites with known identity at 34 °C (Online Resource 4), indicating more pronounced metabolic changes with growth at elevated temperatures.Fig. 5

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.