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Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile.

ABSTRACT: 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 [Formula: see text], which in wild-type strains are modified with mcm5s2U, partially suppress phenotypes of an elp3? strain.To identify metabolic alterations in an elp3? strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified [Formula: see text].Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3? 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 [Formula: see text]. The temperature sensitive elp3? strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions.Culturing an elp3? strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3? strain carrying the wild-type ELP3 gene. Overexpression of hypomodified [Formula: see text] suppressed a subset of the metabolic alterations observed in the elp3? strain.Our results suggest that the presence of ncm5- and mcm5-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

SUBMITTER: Karlsborn T

PROVIDER: S-EPMC5037161 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile.

Karlsborn Tony T Mahmud A K M Firoj AKMF Tükenmez Hasan H Byström Anders S AS Byström Anders S AS

Metabolomics : Official journal of the Metabolomic Society 20160927 12

<h4>Introduction</h4>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 [Formula: see text], which in wild-type strains are modi ...[more]

PMID: 27738410

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Project description:Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA). The transcriptional profile of PKO cells was similar to rho0 mtDNA deleted cells, with perturbations in cholesterol (FDR = 6.35 x 10-13), lipid (FDR = 3.21 x 10-11), and secondary alcohol (FDR = 1.04x10-12) metabolic pathway gene expression compared to wild type parental (TM6) MEFs. Transcriptome analysis indicates processes related to axonogenesis (FDR = 4.49 x 10-3), axon development (FDR = 4.74 x 10-3), and axonal guidance (FDR = 4.74 x 10-3) were overrepresented in PKO cells, consistent with previous studies detailing causative PNPase mutations in delayed myelination, hearing loss, encephalomyopathy, and chorioretinal defects in humans. Overrepresentation analysis revealed alterations in metabolic pathways in both PKO and rho0 cells. Therefore, we assessed the correlation of genes implicated in cell cycle progression and total metabolism and observed a strong positive correlation between PKO cells and rho0 MEFs compared to TM6 MEFs. We quantified the normalized biomass accumulation rate of PKO clones at 1.7% (SD ± 2.0%) and 2.4% (SD ± 1.6%) per hour, which was lower than TM6 cells at 3.3% (SD ± 3.5%) per hour. Furthermore, PKO in mouse inner ear hair cells caused progressive hearing loss that parallels human familial hearing loss previously linked to mutations in PNPase. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.

Dataset Information

Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile.

Publications

Loss of ncm<sup>5</sup> and mcm<sup>5</sup> wobble uridine side chains results in an altered metabolic profile.

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