Cell type-specific regulation of mitochondrial tRNA abundance

To investigate transcript-wide regulation of mitochondrial tRNA stoichiometry and RNA modifications in cells harboring the MERRF mutation, we used demethylase-thermostable group II intron RT tRNA sequencing (DM-tRNA-seq)17. This method uses a highly processive thermostable group II intron reverse-transcriptase (TGIRT) coupled with two demethylases, to overcome the stable secondary structure and Watson–Crick face methylations of tRNAs to obtain quantitative genome-wide information on cellular tRNA populations and their modifications. We prepared tRNA libraries from deacylated RNA isolated from wild-type myoblasts and myoblasts homoplasmic for the MERRF mutation, with or without demethylase treatment. The demethylase-treated samples generated longer reads, which greatly facilitated specific tRNA assignment and quantitation, whereas the untreated samples allowed identification and quantitative comparison of many modifications at the Watson–Crick face18. These libraries were sequenced, and tRNAs were mapped to the human nuclear and mitochondrial genomes (16–40 million total reads per sample, 6–13 million uniquely mapped per sample). Between 12.1–13.4% of all mapped tRNA reads corresponded to mitochondrially encoded tRNAs. The data were highly reproducible among three replicate experiments for both wild-type and homoplasmic mutant cells (Pearson’s coefficient > 0.992 for all pairs of replicates; Supplementary Figure 1A). 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).

The mode of mtDNA transcription can vary across human cell types (so far not studied in myoblasts) and appears to be biased towards nascent RNA synthesis from the promoter of the light coding strand of the genome (Fig. 1a)19. We compared the steady-state abundance of each tRNA with the gene position along the mitochondrial coding strands (heavy and light (Fig. 1a)), but observed no trends for either strand (Fig. 1e). Within the mitochondrial genome, the genes encoding tRNAPhe and tRNAVal flank the 12 S rRNA gene (Fig. 1a), and are transcribed together, along with the 16 S rRNA, as a short polycistronic RNA at a level 1.4–8.8-fold greater than genes downstream along the H-strand depending upon the cell type19. In HeLa cells, the steady-state levels of tRNAPhe and tRNAVal were 2–3-fold higher compared to those of other H-strand tRNAs when determined by quantitative northern blotting20 and concordant with nascent RNA synthesis of these genes for this cell type. This difference was not observed in myoblasts using DM-tRNA-seq. To test whether the discrepancy in the steady-state tRNA abundance may be attributable to methodological or actual biological differences in tRNA regulation between diploid and aneuploid cells, we analyzed mitochondrial tRNA stoichiometry in a high-throughput tRNA sequencing dataset obtained from HEK293T cells using DM-tRNA-seq17. This analysis revealed that, as in HeLa cells, the levels of tRNAPhe and tRNAVal in HEK293T were 2–3-fold higher than those of other H-strand tRNAs (Fig. 1f, g). Taken together, our data suggest differential regulation of the mitochondrial genome in proliferating human cultured cells, highlighting the need to use diploid cells to investigate the pathogenic mechanisms of human mitochondrial gene expression disorders.

The m1A58 modification in tRNALys is absent in myoblasts with the MERRF mutation

Post-transcriptional modifications of mitochondrial tRNAs are functionally important to achieve and stabilize the cloverleaf structure, and for decoding during translation elongation21. In MERRF, the 5-taurinomethyl 2-thiouridine modification on the anticodon wobble base is missing on the tRNALys purified from cybrid cells7 and from a patient liver biopsy22. In the absence of this U34 modification, translation on cognate codons is impaired using an in vitro5 assay. However, metabolic labeling of mitochondrial protein synthesis in human cells is at odds (Fig. 1c) with the in vitro data. Furthermore, lack of the analogous 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) U34 modification of the yeast cytoplasmic tRNALys does not prevent decoding but rather substantially slows down the rate of translation elongation due to ribosome pausing on lysine codons10,11. Therefore, we asked whether other RNA modifications were important to the molecular pathogenesis either directly on tRNALys or to the entire pool of mitochondrial tRNAs as part of regulatory feedback from impaired protein synthesis.

DM-tRNA-seq allowed us to assess quantitatively differences in specific RNA modifications across all mitochondrial tRNAs between wild-type and MERRF-mutant cells. At Watson–Crick face methylation sites, such as N1-methyladenosine (m1A), the thermophilic RT generates both stop and read-through mutation sequencing reads. We used the term “modification index” (MI) to quantify the mutation and stop fractions at each nucleotide position18. (Please see the Methods section for a discussion comparing the sensitivity and accuracy of DM-tRNA-seq vs. traditional mass-spectrometry approaches for detecting RNA modifications).

First, we addressed the abundance of the τm5s2U anticodon wobble base modification of tRNALys between wild-type and MERRF. As expected, we detected a significant decrease in the modification at this position in MERRF samples (Fig. 2a, b), but it was not completely absent. Unlike N1-methyladenosine (m1A), for which the MI value can be used to approximate the modified fraction, the MI of 0.2 for τm5s2U in wild-type cells likely under-represents the modified fraction because the Watson–Crick face perturbation of τm5s2U is 2-thio, which weakens, but does not block, base pairing between the template and dNTP during cDNA synthesis. Nevertheless, the markedly reduced MI in the MERRF sample was consistent with a reduction of the τm5s2U modification. Anticodon wobble base modifications in other mitochondrial tRNAs were also detected in our sequencing reads, but did not differ between wild-type and MERRF cells (Fig. 2b).

Fig. 2 Absence of an m1A modification in the m.8344 A > G tRNALys. a Position sequencing plots of tRNALys from human myoblasts homoplasmic for wild-type or the m.8344 A > G MERRF mutation without (left) and with (right) demethylase treatment. M1A9 produces two peaks, the first corresponds to RT stop and mutations upon copying the m1A nucleotide and the second stop was due to m1A9 present in the RT template. b Quantitative comparisons of U34 anticodon wobble base modifications in mitochondrial tRNAs from human myoblasts. Data represent mean + /− S.D. (n = 3). Paired sample t test. c Quantification of m1A modifications at the indicated positions in mitochondrial tRNAs from human myoblasts. Data represent mean + /− S.D. (n = 3) Full size image

Next, we investigated whether there were any differences in the known m1A modifications. To our surprise, the m1A at position 58 of tRNALys (Fig. 1b) was absent in MERRF cells (Fig. 2a, c). It is important to note that mitochondrial tRNAs do not possess classical D and T arms 9so the actual position of the m1A in mitochondrial tRNALys is nucleotide 54 (m.8348 in the genome), however, we kept with conventional tRNA numbering for this modification. The m1A58 is catalyzed by the methyltransferase ‘writer’ TRMT61B23 and our quantitation of the modification by DM-tRNA-seq in wild-type cells is consistent with previous reports by primer extension and mass-spectrometry23,24, which demonstrate that this methyl modification was present only in a fraction of tRNALys. In contrast, the m1A9 on the D arm required for the formation of the cloverleaf structure of tRNALys25 and catalyzed by a different methyltransferase complex26, was detected in both wild-type and MERRF cells. To test whether other known m1A58 modifications in mitochondrial tRNAs were affected, we analyzed the m1A58 on tRNAs Leu(UUR) and Ser(UCN), as well as m1A16 in tRNAArg and found no difference between wild-type and MERRF cells (Fig. 2c). Together, these data indicated that the loss of the m1A modification in MERRF cells was restricted to tRNALys at position 58. Our sequencing approach expanded the landscape of MERRF pathogenesis by implicating a novel methyl modification on tRNALys, highlighting the versatility and robustness of DM-tRNA-seq for investigating the regulation of mitochondrial gene expression disorders.

m1A58 tRNALys is linked to the MERRF pathogenesis in human skeletal muscle

To assess the disease relevance of the modifications present in the tRNALys from proliferating myoblasts to the molecular pathogenesis of MERRF, we analyzed mitochondrial tRNAs from skeletal muscle biopsies obtained from a control and MERRF patient. Total RNA was isolated from control and MERRF muscle, and tRNA libraries were prepared for sequencing. From each of the libraries, 22–50 million reads were mapped. The levels of nuclear-encoded tRNAs were strongly correlated between the two samples (Supplementary Figure 1B). By contrast, the steady-state level of tRNALys was ~ 50% lower in MERRF skeletal muscle than in the control (Fig. 3a), similar to our findings in myoblasts (Fig. 1e) (note that our cultured myoblasts originated from a different patient than the skeletal muscle biopsy). In skeletal muscle, the frequency of the MERRF mutation was approximately 80% (Fig. 3b), which is consistent with the threshold mutational load sufficient to cause a biochemical defect in oxidative phosphorylation13.

Fig. 3 Novel RNA modifications of tRNALys linked to MERRF pathogenesis in human skeletal muscle. a Quantification of tRNALys abundance in skeletal muscle. b A position sequencing plot of tRNALys without demethylase treatment from skeletal muscle. c A 3′- position sequencing plot for the frequency of adenylation among mitochondrial tRNAs from skeletal muscle of a control and MERRF patient Full size image

To test the disease relevance of the post-transcriptional modifications on tRNALys to MERRF in skeletal muscle, we analyzed the modification index at positions 9, 34, and 58 (Fig. 1b) between the healthy control and patient. The cloverleaf stabilizing m1A9 modification was found on the majority of tRNALys in both the MERRF patient and control (Fig. 3b). In contrast, the modification fraction of the m1A58 in tRNALys was ~0.3 in control and absent in MERRF (Fig. 3b). This finding is entirely consistent with our data from proliferating myoblasts, strongly suggesting a key role of this modification in the pathogenesis of MERRF. The τm5s2U anticodon wobble base modification at U34 was modestly down in MERRF (Fig. 3b). Unfortunately, our analyses could not distinguish whether the m1A58 co-exists in cis with the τm5s2U anticodon wobble base modification. Together, our findings establish the relevance and expand the landscape of post-transcriptional RNA modifications on tRNALys to the molecular pathogenesis of MERRF.

A recent study demonstrated that aminoacylation of mitochondrial tRNAs bearing a CCA tail is regulated by oligoadenylation, catalyzed by the mitochondrial poly(A) polymerase (PAPD1); the steady-state level of this modification is governed by the balance between the activities of PAPD1 and phosphodiesterase 12 (PDE12), and is particularly prominent for tRNALys and tRNASer2(AGY)15. Whereas another study showed how structurally abnormal mitochondrial tRNAs can be polyadenylated for degradation27. Therefore, we asked whether the adenylation mechanism is relevant to the pathogenesis of MERRF. For this analysis, we sorted sequence reads into two pools based upon the presence or absence of the CCA tail. Aminoacylation of tRNAs requires the addition of the CCA extension, which is catalyzed by a mitochondrially targeted TRNT1, and is critical for mitochondrial protein synthesis and human health28,29. When we analyzed the tRNA sequencing reads, oligoadenylation was widespread but only among the tRNA pool lacking the CCA extension in both control and MERRF (Fig. 3c). There was differential oligoadenylation between control and MERRF of selected mitochondrial tRNAs, including tRNALys (Fig. 3c). These data indicate that adenylation of mitochondrial tRNAs is a far more complex process than previously thought and may play a role in the regulation of mitochondrial tRNA populations and the pathogenesis of human disease.

tRNALys RNA modifications have specific effects on translation

Since the m1A58 modification on tRNALys is consistently absent in MERRF myoblasts and skeletal muscle, we asked whether re-establishing the modification in the MERRF myoblasts could modulate mitochondrial translation. To this end, we retrovirally transduced myoblasts with the TRMT61B cDNA, reasoning that overexpression of this key methyltransferase would restore the modification on the tRNALys. Indeed, TRMT61B overexpression restored the m1A modification in MERRF tRNALys (Fig. 4), but did not alter the steady-state abundance of tRNALys (Supplementary Figure 2A). Importantly, TRMT61B overexpression increased the synthesis of selected proteins and suppressed the generation of aberrantly sized polypeptides (Fig. 5a, b; Supplementary Figure 2B). The magnitude of this effect did not correlate with the number of lysine codons of a given polypeptide. (Supplementary Figure 2C).

Fig. 4 Restoration of the m1A58 modification in tRNALys. a Schematic of the primer extension assay for genotyping m1A58 in tRNALys. b A representative primer extension analysis on total RNA from human myoblasts stably transduced by retrovirus with the indicated cDNAs. c Quantification of primer extension analysis from two independent experiments. Proportion of m1A58 was calculated as follows (m1A 8348 / (m1A 8348 + G 8342)) or (m1A 8348 / (m1A 8348 + G 8344)) Full size image

Fig. 5 tRNALys RNA modifications reveal specific effects to translation fidelity. a A representative 35S pulse (30 min) metabolic labeling into mitochondrial protein synthesis of human myoblasts stably homoplasmic for the indicated mitochondrial DNA transduced by retrovirus with the indicated cDNAs. Aberrantly sized labeled polypeptides are indicated. b Quantification of 35S incorporation into selected mitochondrial proteins during a 30 min pulse (from a). Data are represented as mean + /− S.D. from three biological experiments. c A representative 24 and 48 h cold chase following a 30 min 35S pulse metabolic labeling of mitochondrial protein synthesis in human myoblasts. d Quantification of MT-ATP6 stability in the chase relative to wild-type cells transduced with an empty vector. Data is mean + /− S.D. from three biological experiments except for MTO1 at 48 h, where only the data from two independent experiments are shown. e Immunoblotting of whole-cell lysates from human myoblasts homoplasmic for the indicated mitochondrial DNA transduced by retrovirus with the indicated cDNAs decorated with the indicated antibodies. Representative data of multiple independent experiments Full size image

Next, we asked whether the increased protein synthesis with TRMT61B overexpression also modulated the short term and long-term stability of mitochondrial nascent chains. Even though there was a 2-fold increase in 35S-incorporation in MT-CO1 and MT-ATP6 (Fig. 5b), these proteins were still unstable but with differential effects (Fig. 5c-e). By contrast, TRMT61B overexpression in the wild-type background led to a two-fold increase of the m1A58 on tRNALys (Fig. 4c) and a selective dominant-negative effect specific to MT-CO1 nascent chain synthesis and long-term stability (Fig. 5a, b, e). Recent reports have also documented specific m1A modifications in mitochondrial 12 S and 16 S rRNA, and mRNAs, including MT-CO130,31,32. The m1A modification at position 947 of the 16 S rRNA was proposed to be important for human mitochondrial ribosome subunit stability and is catalyzed by TRMT61B30. To test if mitochondrial ribosome stability was affected by TRMT61B overexpression, we assessed the steady-state levels of mitochondrial ribosomal proteins from the small and large subunit by immunoblotting because their abundance is a reliable proxy for stability33 but found no difference (Fig. 5e). Our data showed that restoration of the m1A58 on tRNALys can enhance translation in MERRF, arguing against this methyl modification as being a strictly negative regulator of mitochondrial gene expression as has been proposed for the m1A modification on mitochondrial mRNAs31,32. Rather, there appears to be a tight regulation of this methyl modification on mitochondrial RNAs to finely tune mitochondrial protein synthesis.

Because reduced modification of the anticodon wobble base position is implicated in MERRF pathogenesis, we wanted to test whether overexpression of the key enzymes could also modulate mitochondrial translation. MTU1 catalyzes the thiol modification of the anticodon wobble base, a reaction that is required for human health34, but appears to have no effect on mitochondrial protein synthesis in human fibroblasts35. Consistent with this finding, overexpression of MTU1 in wild-type and MERRF myoblasts had no effect on mitochondrial protein synthesis (Supplementary Figure 2D and 2E). In contrast, overexpression of MTO1 had a marked effect on mitochondrial protein synthesis and protein stability (Fig. 5). MTO1 forms a complex with the small GTPase GTPBP3 to catalyze the 5-methylaminomethyl (mnm5U34) modification on the anticodon wobble base found in mitochondrial tRNAs of Glu, Lys, Gln, Leu(UUR), and Trp9. MTO1 overexpression restored mitochondrial protein synthesis to wild-type levels (Fig. 5a, b), but there was a differential effect on the stability of individual proteins (Fig. 5c–e) that did not correlate with the abundance of lysine codons in a given polypeptide (Supplementary Figure 2C). Moreover, these effects were independent of the m1A58 modification (Fig. 4b, c). Although the rate of MT-CO1 synthesis increased, these nascent chains remained unstable in contrast to MT-ATP6. No adverse effects were observed on wild-type myoblasts overexpressing MTO1. Collectively, these findings demonstrate that post-transcriptional modifications of mitochondrial tRNAs exert differential effects on protein synthesis and stability, revealing the profound influence that RNA modifications have on mitochondrial gene expression and the pathogenesis of human disease.