Abstract
Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N1-methyladenosine (m1A) modification is missing at position 58 in the mitochondrial tRNALys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNALys, we demonstrated the importance of the m1A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.
Highlights
Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood
Mitochondria contain a unique set of ribosomes and tRNAs dedicated to the synthesis of the 13 proteins encoded in mitochondrial DNA (Fig. 1a), all of which are essential for aerobic energy metabolism
The abundance of mitochondrial tRNALys was significantly reduced in MERRF cells (Fig. 1d, e), whereas the steady-state levels of the other 21 mitochondrial tRNAs were essentially identical between wild-type and MERRF cells (Fig. 1e)
Summary
Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. A > G in tRNALys, first identified >25 years ago in patients with MERRF (myoclonus epilepsy, ragged-red fibers) syndrome3(Fig. 1b) This mutation causes a severe translation defect during mitochondrial protein synthesis, which has been attributed to a decrease in the steadystate abundance of the mitochondrial tRNALys, reduced aminoacylation of tRNALys, and lack of a post-transcriptional 5taurinomethyl 2-thiouridine RNA modification of the anticodon wobble base[4,5,6,7]. TRNA stoichiometry and RNA modifications are critical regulators of protein synthesis[16], but the molecular basis by which mitochondrial gene expression is disrupted with this pathogenic tRNALys mutation associated with MERRF remains unclear To bridge this gap, we capitalized on a methodological advance in next-generation RNA sequencing to investigate the consequences of the MERRF tRNALys mutation on the stoichiometry and modifications of the entire mitochondrial tRNA pool in affected patient cells and tissues. Our results implicate a novel tRNA modification in the pathogenesis of MERRF and reveal an extensive and unexpected influence of RNA modifications on the regulation and fidelity of mitochondrial gene expression
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