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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.


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Score plots summarizing the PLS-DA modelling of strains grown at 34 °C. a PLS-DA score plot when modelling the elp3Δ strain with an empty low copy pRS315 vector (elp3Δ-l.c.-empty) against the elp3Δ strain containing the wild-type ELP3 gene on a pRS315 vector (elp3Δ-l.c.-ELP3). b Random permutation (20 randomizations) test-validation plot of PLS-DA model in a. c PLS-DA score plot when modelling the elp3Δ strain with an empty high copy pRS425 vector (elp3Δ-h.c.-empty) against the elp3Δ strain containing a pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE). d Random permutation (20 randomizations) test-validation plot of PLS-DA model in (c). Each dot in a, c and e represents a technical replicate from three different biological replicates
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Fig4: Score plots summarizing the PLS-DA modelling of strains grown at 34 °C. a PLS-DA score plot when modelling the elp3Δ strain with an empty low copy pRS315 vector (elp3Δ-l.c.-empty) against the elp3Δ strain containing the wild-type ELP3 gene on a pRS315 vector (elp3Δ-l.c.-ELP3). b Random permutation (20 randomizations) test-validation plot of PLS-DA model in a. c PLS-DA score plot when modelling the elp3Δ strain with an empty high copy pRS425 vector (elp3Δ-h.c.-empty) against the elp3Δ strain containing a pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE). d Random permutation (20 randomizations) test-validation plot of PLS-DA model in (c). Each dot in a, c and e represents a technical replicate from three different biological replicates

Mentions: We looked for alterations in levels of specific metabolites in the elp3Δ strain using partial least squares regression discriminant analysis (PLS-DA) (Figs. 3a–f, 4a–d). Metabolites with a variable importance for the projection (VIP) below one were excluded (Chong and Jun 2005). Briefly, PLS-DA allows analysis of large sample sets structured in the form of classes. The classes are separated according to a comparison between all variables within one class and all variables within another class, and subsequent prediction of the variables that account for the class separation. Variables that are good predictors for separating one class from another have a high VIP score, while variables with a low VIP score do not contribute to class separation.Fig. 3


Loss of ncm 5 and mcm 5 wobble uridine side chains results in an altered metabolic profile
Score plots summarizing the PLS-DA modelling of strains grown at 34 °C. a PLS-DA score plot when modelling the elp3Δ strain with an empty low copy pRS315 vector (elp3Δ-l.c.-empty) against the elp3Δ strain containing the wild-type ELP3 gene on a pRS315 vector (elp3Δ-l.c.-ELP3). b Random permutation (20 randomizations) test-validation plot of PLS-DA model in a. c PLS-DA score plot when modelling the elp3Δ strain with an empty high copy pRS425 vector (elp3Δ-h.c.-empty) against the elp3Δ strain containing a pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE). d Random permutation (20 randomizations) test-validation plot of PLS-DA model in (c). Each dot in a, c and e represents a technical replicate from three different biological replicates
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Fig4: Score plots summarizing the PLS-DA modelling of strains grown at 34 °C. a PLS-DA score plot when modelling the elp3Δ strain with an empty low copy pRS315 vector (elp3Δ-l.c.-empty) against the elp3Δ strain containing the wild-type ELP3 gene on a pRS315 vector (elp3Δ-l.c.-ELP3). b Random permutation (20 randomizations) test-validation plot of PLS-DA model in a. c PLS-DA score plot when modelling the elp3Δ strain with an empty high copy pRS425 vector (elp3Δ-h.c.-empty) against the elp3Δ strain containing a pRS425 vector carrying the tRNA genes tK(UUU), tQ(UUG) and tE(UUC) (elp3Δ-h.c.-tKQE). d Random permutation (20 randomizations) test-validation plot of PLS-DA model in (c). Each dot in a, c and e represents a technical replicate from three different biological replicates
Mentions: We looked for alterations in levels of specific metabolites in the elp3Δ strain using partial least squares regression discriminant analysis (PLS-DA) (Figs. 3a–f, 4a–d). Metabolites with a variable importance for the projection (VIP) below one were excluded (Chong and Jun 2005). Briefly, PLS-DA allows analysis of large sample sets structured in the form of classes. The classes are separated according to a comparison between all variables within one class and all variables within another class, and subsequent prediction of the variables that account for the class separation. Variables that are good predictors for separating one class from another have a high VIP score, while variables with a low VIP score do not contribute to class separation.Fig. 3

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