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Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy

ABSTRACT: Editing of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs) is of high significance for protein homeostasis, whose impairment causes neurodegeneration. However, whether mitochondrial translation needs fidelity and the significance of proofreading (editing) by mitochondrial aaRSs are long-term mysteries. Here, we showed that NIH-3T3 cell line critically depend on the editing of mitochondrial threonyl-tRNA synthetase (Tars2) editing, the disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest at G0/G1 phase. ROS removal by N-acetylcysteine relieved abnormal cell proliferation.

ORGANISM(S): Mus musculus

PROVIDER: GSE232410 | GEO | 2023/08/25

REPOSITORIES: GEO

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The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation

Project description:Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here, we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinations for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.

2021-12-04 | GSE185015 | GEO

The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation

2021-12-04 | GSE174448 | GEO

Saccharomyces cerevisiae translational fidelity

Project description:Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that a C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, directed-evolution of the C268A mutant result in intragenic mutations that restore heat resistance but not editing. C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, and the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in ThrRS aminoacylation or editing alone is not sufficient to cause heat sensitivity, and that C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

2024-01-26 | PXD042145 | Pride

On the mechanism and origin of isoleucyl-tRNA synthetase editing against norvaline

Project description:Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared the propensity of norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), aminoacylation and pre-transfer editing with Nva and Val occur at similar rates. Post-transfer editing was, however, more efficient with Nva as IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS post-transfer editing domain being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. Thus, we hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins.

2019-01-28 | PXD011051 | Pride

YAMAT-seq of mature tRNA pools in the bacterium Pseudomonas fluorescens SBW25 and derivatives

Project description:Mature tRNA pools were measured using an adaptation of Y-shaped Adapter-ligated MAture tRNA sequencing (YAMAT-seq) (Shigematsu et al., 2017) in nine strains derived from the model bacterium, Pseudomonas fluorescens SBW25. The aim of the experiment was to determine the effect on the mature tRNA pool of (i) removing the single-copy serCGA gene by genetic engineering, and (ii) duplicating the serTGA gene during a subsequent, compensatory evolution experiment. We found that (i) results in the loss of tRNA-Ser(CGA) from the mature tRNA pool, and (ii) results in a 2-fold higher expression of tRNA-Ser(UGA). tRNA-Ser(UGA) presumably substitutes for tRNA-Ser(CGA) by wobble base pairing to translate codon 5'-UCG-3'.

2020-11-02 | GSE144791 | GEO

Tarsl2 is evolved not for tRNA charging in vivo

Project description:As a newly evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase), TARSL2 (TARS3) encodes a protein highly similar with TARS1 with only obvious difference in N-terminus. Albeit with tRNA charging activity in vitro and incorporated into the multiple tRNA synthetase complex (MSC), whether TARSL2 is able to function as a tRNA synthetase to substitute TARS1 in vivo is unclear. In this work, we found that Tars1 was essential for embryonic development. In contrast, knockout of Tarsl2 was achievable. Loss of Tarsl2 had no effect on both abundance and charging levels of all tRNAThr isoacceptors, indicating Tarsl2 is not for tRNAThr metabolism. Furthermore, Tarsl2 deletion did not influence protein levels of other MSC components, MSC integrity and aminoacylation levels of non-cognate tRNAs by MSC components. However, a severe retardation of mice development was observed after 3 weeks accompanied with an elevated metabolism capacity and muscle development abnormity. We further constructed a Tarsl2-deleted zebrafish, which exhibited no change in the level and charging of tRNAThrs. Collectively, these data suggest that Tarsl2 is evolved not for canonical tRNAThr aminoacylation and cannot substitute Tars1 in vivo.

2023-05-31 | GSE218318 | GEO

synthetic metagenome

Project description:Dissecting primer editing by proofreading polymerases

| PRJNA673973 | ENA

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs

Project description:Mitochondria contain a specific translation machinery for the synthesis of respiratory chain components encoded on the mitochondrial genome. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial genome and, similar to their cytoplasmic counterparts, are modified at various positions. Here, we find that the RNA methyltransferase METTL8, is a mitochondrial protein that facilitates m3C methylation at position C32 of mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knock out cells show reduced and over expressing cells enhanced respiratory chain activity. In pancreatic cancer, METTL8 levels are high, which correlates with patient survival. Indeed, METTL8 up regulation stimulates respiratory chain activity in these cells. Ribosome occupancy analysis using ribosome profiling revealed ribosome stalling on mt-tRNASer(UCN) and mt-tRNAThr codons and mass spectrometry analysis of native ribosomal subcomplexes unraveled reduced respiratory chain incorporation of the mitochondria encoded proteins ND6 and ND1. A well-balanced translation of mt-tRNASer(UCN) and mt-tRNAThr codons through METTL8-mediated C32 methylation might therefore provide optimal respiratory chain compositions and function.

2023-01-02 | GSE180400 | GEO

METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

Project description:A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear-encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.

2022-05-19 | PXD030418 | Pride

tRNAs marked with CCACCA are targeted for degradation

Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included

2011-11-17 | E-GEOD-33756 | biostudies-arrayexpress

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