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

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 determinants 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.

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

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.

Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

Mitochondrial tRNA Mutations Associated With Essential Hypertension: From Molecular Genetics to Function

Essential hypertension (EH) is one of the most common cardiovascular diseases worldwide, entailing a high level of morbidity. EH is a multifactorial disease influenced by both genetic and environmental factors, including mitochondrial DNA (mtDNA) genotype. Previous studies identified mtDNA mutations that are associated with maternally inherited hypertension, including tRNAIle m.4263A>G, m.4291T>C, m.4295A>G, tRNAMet m.4435A>G, tRNAAla m.5655A>G, and tRNAMet/tRNAGln m.4401A>G, et al. These mtDNA mutations alter tRNA structure, thereby leading to metabolic disorders. Metabolic defects associated with mitochondrial tRNAs affect protein synthesis, cause oxidative phosphorylation defects, reduced ATP synthesis, and increase production of reactive oxygen species. In this review we discuss known mutations of tRNA genes encoded by mtDNA and the potential mechanisms by which these mutations may contribute to hypertension.

Hopeful monsters: unintended sequencing of famously malformed mite mitochondrial tRNAs reveals widespread expression and processing of sense–antisense pairs

Although tRNA structure is one of the most conserved and recognizable shapes in molecular biology, aberrant tRNAs are frequently found in the mitochondrial genomes of metazoans. The extremely degenerate structures of several mitochondrial tRNAs (mt-tRNAs) have led to doubts about their expression and function. Mites from the arachnid superorder Acariformes are predicted to have some of the shortest mt-tRNAs, with a complete loss of cloverleaf-like shape. While performing mitochondrial isolations and recently developed tRNA-seq methods in plant tissue, we inadvertently sequenced the mt-tRNAs from a common plant pest, the acariform mite Tetranychus urticae, to a high enough coverage to detect all previously annotated T. urticae tRNA regions. The results not only confirm expression, CCA-tailing and post-transcriptional base modification of these highly divergent tRNAs, but also revealed paired sense and antisense expression of multiple T. urticae mt-tRNAs. Mirrored expression of mt-tRNA genes has been hypothesized but not previously demonstrated to be common in any system. We discuss the functional roles that these divergent tRNAs could have as both decoding molecules in translation and processing signals in transcript maturation pathways, as well as how sense–antisense pairs add another dimension to the bizarre tRNA biology of mitochondrial genomes.

Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure-Function Map

The evolution of tRNA multi-gene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified orthologies to compare substitution rates across tRNA bodies at single-site resolution. All rapidly evolving sites fell within the same metal-ion-binding pocket at the interface of the two major stacked helical domains. We applied our tRNA Structure-Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica. We found that, although predicted tRNA structure-function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidlyevolving ion-binding pocket — Cytosine 17 (C17), ancestrally informative for lysylation identity — independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly at least three times, in evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in nearby heterologous gene clusters, suggesting that heterologous gene conversion as well as structural similarities of the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs of the D. ananassae lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure-function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multi-gene families.

7-Methylguanosine Modifications in Transfer RNA (tRNA)

More than 90 different modified nucleosides have been identified in tRNA. Among the tRNA modifications, the 7-methylguanosine (m7G) modification is found widely in eubacteria, eukaryotes, and a few archaea. In most cases, the m7G modification occurs at position 46 in the variable region and is a product of tRNA (m7G46) methyltransferase. The m7G46 modification forms a tertiary base pair with C13-G22, and stabilizes the tRNA structure. A reaction mechanism for eubacterial tRNA m7G methyltransferase has been proposed based on the results of biochemical, bioinformatic, and structural studies. However, an experimentally determined mechanism of methyl-transfer remains to be ascertained. The physiological functions of m7G46 in tRNA have started to be determined over the past decade. For example, tRNA m7G46 or tRNA (m7G46) methyltransferase controls the amount of other tRNA modifications in thermophilic bacteria, contributes to the pathogenic infectivity, and is also associated with several diseases. In this review, information of tRNA m7G modifications and tRNA m7G methyltransferases is summarized and the differences in reaction mechanism between tRNA m7G methyltransferase and rRNA or mRNA m7G methylation enzyme are discussed.

7-Methylguanosine Modifications in tRNA

More than 90 different modified nucleosides have been identified in tRNA. Among the tRNA modifications, the 7-methylguanosine (m7G) modification is found widely in eubacteria, eukaryotes, and a few archaea. In most cases, the m7G modification occurs at position 46 in the variable region and is a product of tRNA (m7G46) methyltransferase. The m7G46 modification forms a tertiary base pair with C13-G22, and stabilizes the tRNA structure. Recently, we have proposed a reaction mechanism for eubacterial tRNA m7G methyltransferase (TrmB) based on the results of biochemical studies and previous biochemical, bioinformatic, and structural studies by others. However, an experimentally determined mechanism of methyl-transfer remains to be ascertained. The physiological functions of m7G46 in tRNA have started to be determined over the past decade. To be able to better respond to diseases and infections in which the m7G modification is considered to be involved, it is still necessary to further understand the catalytic mechanism of AdoMet and/or the tRNA bound form of m7G methyltransferases. In this review, information of tRNA m7G modifications and tRNA m7G methyltransferases are summarized and the differences in reaction mechanism between tRNA m7G methyltransferase and rRNA or mRNA m7G methylation enzyme are discussed.