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NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE84664 | GEO | 2016/08/09

SECONDARY ACCESSION(S): PRJNA330795

REPOSITORIES: GEO

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NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation

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

Homo sapiens

Project description:NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation

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A Quantitative Sequencing Method for 5-Formylcytosine in RNA

Project description:5-Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt-tRNAMet) and cytosolic leucine tRNA (ct-tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in mRNA of yeast and human cells, but its transcriptome-wide distribution has not been studied. Here we report f5C-seq, a quantitative sequencing method to map f5C transcriptome-wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pico-brane, and DHU can be exclusively read as U during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C-to-U mutation signature. We validated f5C-seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1-depleted cells. By applying f5C-seq to small RNA, we identified 13 and 11 new f5C sites in HeLa and mESC tRNA, respectively.

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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.

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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.

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

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endogenous mRNA decay in 293t, hela, RPE cell line

Project description:mRNA endogenous decay profiles approach in 293T, HeLa and RPE cells. mRNA translation decodes nucleotide into amino acid sequences. However, translation has also been shown to affect mRNA stability depending on codon composition in model organisms, although universality of this mechanism remains unclear. Here, using three independent approaches to measure exogenous and endogenous mRNA decay, we define which codons are associated with stable or unstable mRNAs in human cells. We demonstrate that the regulatory information affecting mRNA stability is encoded in codons and not in nucleotides. Stabilizing codons tend to be associated with higher tRNA levels and higher charged/total tRNA ratios. While mRNAs enriched in destabilizing codons tend to possess shorter poly(A)-tails, the poly(A)-tail is not required for the codon-mediated mRNA stability. This mechanism depends on translation; however, the number of ribosome loads into a mRNA modulates the codon-mediated effects on gene expression. This work provides definitive evidence that translation strongly affects mRNA stability in a codon-dependent manner in human cells.

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