Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology

Abstract

SUMMARYHuman mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that theN1-methylation of guanosine at position 9 (m1G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m1G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that theN1-methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

Unraveling RNA Conformation Dynamics in Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episode Syndrome with Solid-State Nanopores

“…In a separate study, we have provided nanopore data showing that the m 1 G9modified WT tRNA transcript has a structure that closely resembles the native structure of the bovine tRNA counterpart, which shares close sequence identity with human mt-Leu(UAA). 83 This similarity indicates that single m 1 G9 methylation is sufficient to fold the WT tRNA into a nearnative structure. Nonetheless, we do not yet know whether the WT and the variant tRNA would have a similar structure that differs in stability and/or compactness or whether they would have a fundamentally distinct structure.…”

“…Notably, while the nanopore-based approach can discern tRNA conformational changes due to a single-nucleotide substitution or due to the addition or removal of a single methyl group, it does not provide the actual structure of the WT or the A14G variant of mt-Leu­(UAA). In a separate study, we have provided nanopore data showing that the m 1 G9-modified WT tRNA transcript has a structure that closely resembles the native structure of the bovine tRNA counterpart, which shares close sequence identity with human mt-Leu­(UAA) . This similarity indicates that single m 1 G9 methylation is sufficient to fold the WT tRNA into a near-native structure.…”

“…86 Modified versions of the same tRNAs were prepared by ligating a chemically synthesized 18-nucleotide 5′-RNA fragment to a 60-mer 3′-RNA fragment prepared by in vitro transcription. 83 By including GMP in the transcription reaction, most of the RNAs contained a 5′-phosphate. Ligation reactions were carried out in the absence of a splint by incubating equimolar amounts of the 18-mer and 60-mer RNAs with T4 Rnl2 and ATP for 1 h at 37 °C.…”

“…Since the 5′-end of these tRNAs did not contain two guanosine residues, they were transcribed with an upstream self-cleaving hammerhead ribozyme to generate mature tRNAs with a 5′-hydroxyl group . Modified versions of the same tRNAs were prepared by ligating a chemically synthesized 18-nucleotide 5′-RNA fragment to a 60-mer 3′-RNA fragment prepared by in vitro transcription . By including GMP in the transcription reaction, most of the RNAs contained a 5′-phosphate.…”