Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA330795 | ENA |
REPOSITORIES: ENA
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Project description:Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilization of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. HEK293 cell lines expressing His-FLAG-tagged NSUN3 or the His-FLAG tag alone were crosslinked using UV or treated with 5-azacytidine and analysed by CRAC
2016-08-09 | E-GEOD-84664 | biostudies-arrayexpress
Project description:Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilization of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.
2016-08-09 | GSE84664 | GEO
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
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
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 | GSE174448 | GEO
Project description:In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation, and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain - an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly-structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.
2019-08-10 | GSE133315 | GEO
Project description:5-Formylcytidine (f5C) is one type of post-transcriptional RNA modifi-cations, which is known at the wobble position of tRNA in mitochon-dria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil (DHU), thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. Thousands of f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA. Reference build: S288C_reference_genome_R64-2-1_20150113
2021-12-01 | GSE133138 | GEO
Project description:Methyltransferase MRM2 methylates the 2’-hydroxyl group of ribonucleotide U1369 of human 16S mt-rRNA. Mutations in MRM2 have been implicated in human mitochondrial-related disease. This study investigates the role of MRM2 in the biogenesis and function of human mitochondrial ribosomes. Absence of MRM2 leads to a severe defect in mitochondrial translation, with the mtLSU being trapped in immature assembly states.
2021-12-17 | E-MTAB-11292 | biostudies-arrayexpress
Project description:Members of the mammalian AlkB family are known to mediate nucleic acid demethylation. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria (mt) and affects metabolism, but its function and mechanism of action are unknown. Here, we report an approach to site-specifically detect m1A, m3C, m1G, and m22G modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates N2, N2-dimethylguanosine (m22G) and N1-methyladenosine (m1A) within mt-Ile and mt-Leu1 pre-tRNA regions, respectively, in nascent polycistronic mt-RNA. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mt-RNAs. Depletion of ALKBH7 leads to increased polycistronic mt-RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, as well as notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mt-RNA processing and mitochondrial activity.
2021-06-02 | GSE148202 | GEO
Project description:Mitochondria are organelles that generate most of the energy in eukaryotic cells in the form of ATP via oxidative phosphorylation in eukaryote. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA are required to translate essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. These modifications are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. We performed a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species, and revised the modification status of some of the previously studied species. In total, we found 17 kinds of RNA modifications at 137 positions (8.7% in 1,575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for human mt-tRNA modifications are provided. We here demonstrated that both QTRT1 and QTRT2 are required for biogenesis of queuosine (Q) at position 34 of four mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the mitochondrial decoding system, and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.
2020-08-28 | GSE150439 | GEO