Mitochondrial translation requires folate-dependent tRNA methylation

Abstract

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine1,2,3. Antifolates are important immunosuppressive4 and anticancer agents5. In proliferating lymphocytes6 and human cancers7,8, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism2,9. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

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Figure 1: Mitochondrial respiratory chain function is dependent on SHMT2 catalytic activity.
Figure 2: SHMT2-knockout-induced respiratory chain deficiency is caused by mitochondrial methylene-THF depletion but is unrelated to dTTP synthesis.
Figure 3: Mitochondrial ribosome profiling reveals that SHMT2-knockout cells are deficient in translating specific guanosine-ending codons.
Figure 4: MTO1/GTPBP3-dependent tRNA methylation requires mitochondrial methylene-THF.

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Acknowledgements

We thank T. Pan, W. Lu, L. Chen, L. Parsons, W. Wang and T. Srikumar, and all members of the Rabinowitz laboratory. This work was supported by funding to J.D.R. from the US National Institutes of Health (NIH) (R01CA163591 and DP1DK113643) and StandUp to Cancer (SU2C-AACR-DT-20-16). G.S.D. was supported by a postdoctoral fellowship (PF-15-190-01- TBE) from the American Cancer Society. J.A.M. was supported by the science fund of the Paracelsus Medical University Salzburg (E-12/15/076-MAY). Z.G. was supported by the NIH (DP1AI124669).

Author information

R.J.M., G.S.D. and J.D.R. conceived the project and designed the experiments. R.J.M. and J.D.R. wrote the manuscript. R.J.M., G.S.D. and S.H.L performed biochemical experiments. Z.G. and W.S. were involved in study design and data interpretation. W.S. and J.A.M. contributed primary patient cell lines. All authors reviewed and edited the manuscript before submission.

Correspondence to Joshua D. Rabinowitz.

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Competing interests

J.D.R. is a founder of Raze Therapeutics, which aims to target 1C metabolism for cancer therapy.

Additional information

Reviewer Information Nature thanks D. Appling, V. Gohil and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 SHMT2 deletion-induced respiratory chain dysfunction in different cellular backgrounds and clones.

a, Change in media colour after 48 h cell growth. b, c, Lactate secretion (b) and normalized NAD+/NADH ratio (c) of HCT116 knockout cell lines (n = 6). d, e, Basal respiration as measured by Seahorse XF analyser (n = 3) (d) and normalized NAD+/NADH ratio (n = 3) (e) of HEK293T folate 1C gene CRISPR–Cas9 knockout cell lines. f, Normalized levels of TCA cycle and associated metabolites (n = 3). g, Steady-state labelling fraction into citrate from [U-13C]substrates glutamine (left) and glucose (right) (n = 3). h, Immunoblot of extracted mitochondria for subunits of respiratory chain complexes I–V (CI–CV) and markers of mitochondrial mass. i, Mitochondrial complex I levels (NDUFS4) in independent HCT116 folate 1C gene knockout clones. Data are mean ± s.e.m. n indicates the number of biological replicates. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values).

Extended Data Figure 2 Catalytically deficient SHMT2 constructs.

a, Mapping of mutated amino acid residues on human SHMT1 (PDB code 1BJ481) using iCn3D and alignment of E. coli serine hydroxymethyltransferase (GLYA), H. sapiens mitochondrial serine hydroxymethyltransferase 2 (GLYM) and cytosolic serine hydroxymethyltransferase 1 (GLYC). Positions for GLYM are given with reference to GenBank NM_005412.5. b, Sanger sequencing traces of mutant constructs. c, Immunoblot for mitochondrial complex I levels (NDUFS4) in cell lines re-expressing catalytically deficient forms of SHMT2.

Extended Data Figure 3 Restoring SHMT2 catalytic activity normalizes 1C flux, respiratory chain expression, glycolytic activity, and cell growth.

a, Immunoblot of re-expression of catalytically active SHMT2 (left) and the effects of its re-expression on mitochondrial complex I and II levels (right). bf, Effect of re-expression of catalytically active and inactive forms of SHMT2 in two different ΔSHMT2 clones in the HEK293T background. b, Normalized NAD+/NADH ratio (n = 6). c, Lactate secretion and glucose uptake (n = 6). d, Cell proliferation (n = 6). e, Purine biosynthesis intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) levels (n = 4) as an indicator of cytosolic folate 1C status. f, [2,3,3-2H]serine tracing to differentiate cytosolic from mitochondrial folate 1C unit production for incorporation into deoxythymidine triphosphate (n = 3). Data are mean ± s.e.m. n indicates the number of biological replicates. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values). Source data

Extended Data Figure 4 Oxidative phosphorylation defect is caused by a post-transcriptional mechanism independent of methionine formylation.

a, Fraction of initiating amino acid (formylmethionine versus methionine) of mitochondrial-expressed COX1 peptide determined by high-resolution LC–MS (wild type n = 4, ∆SHMT2 n = 3, ΔMTHFD2 n = 2). b, Lactate secretion (n = 3) upon sarcosine supplementation (1 mM). c, Relative mtDNA levels in HEK293T cells (n = 3). d, Agarose gel of mtDNA long-range PCR products of HCT116 and HEK293T knockout cell lines. e, Relative mRNA levels of mtDNA-encoded respiratory chain subunits in the HEK293T background (n = 3). f, Gene expression levels in SHMT2-knockout cell lines compared to SHMT2 wild-type re-expressed lines by total RNA sequencing. Each dot represents mean gene expression as derived from two biological replicates of two independent knockout clones and matched re-expressed lines (n = 4). Genes linked to human OXPHOS function37 are highlighted in red. Significantly differentially expressed genes are listed in Supplementary Table 2. g, Position-dependent next-generation sequencing coverage of mtDNA in HEK293T wild-type, SHMT2-knockout and MTHFD2-knockout cell lines supports the absence of deletions due to SHMT2 loss. h, Corresponding variant position and frequency. Variant list is provided in Supplementary Table 1. Data are mean ± s.e.m. n indicates the number of biological replicates. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values).

Extended Data Figure 5 Impairment of mitochondrial translation due to loss of SHMT2.

a, SDS–PAGE of [35S]methionine-labelled mitochondrially translated proteins in wild-type (lane 1) and two SHMT2-knockout (lane 2 and 3) HEK293T cell lines. Decreased synthesis of COX1 and COX2/3 are evident upon short exposure and reduced synthesis of ND5 and ND6 is more easily visualized upon longer exposure. b, Absorbance at 254 nm upon sucrose gradient fractionation of cell lysates digested by micrococcal nuclease (Fig. 3a). Fractions corresponding to 4 and 5 were collected for mitochondrial ribosome enrichment as shown on the matched immunoblot for mitochondrial ribosome subunit MRPL11. c, Read length distribution (top) and read length-dependent sub-codon read phasing (bottom) across the 13 mitochondrial protein-coding transcripts. Data in c are based on the mitochondrial ribosome profiling experiment in Fig. 3, and represent the mean of two technical replicates of two independent samples.

Extended Data Figure 6 Mitochondrial ribosome stalling at guanosine-ending split codon box nucleotide triplets suggests deficient 5-taurinomethyluridine modification.

a, Expanded version of Fig. 3b, showing the mean cumulative ribosome protected fragments of all mitochondrial protein-coding genes. b, Mean relative density of actively translating (that is, not stalled) ribosomes for mitochondrial transcripts. Data in a and b represent two technical replicates of two independent samples. c, Enzymatic activities of citrate synthase and individual mitochondrial respiratory chain complexes from mitochondrial extracts (n = 5). Data are mean ± s.e.m. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values). d, Mitochondrial genetic code table with split codon boxes depending on taurinomethylated tRNAs for translation highlighted in red. Codons decoded by anticodon formylcytidine-containing tRNAMet are highlighted in blue. e, Mean codon-specific mitochondrial ribosome occupancy of HCT116 SHMT2/MTHFD2 double-knockout cell lines supplemented with sarcosine (1 mM). Codons highlighted in red are decoded by tRNAs carrying a 5-taurinomethyluridine modification. The supplementation with sarcosine prevents the stalling normally observed with SHMT2 deletion (n = 2). Source data

Extended Data Figure 7 tRNA modification status in ∆SHMT2 and effects of 5-taurinomethyluridine modification loss caused by human disease gene MTO1.

a, Total ion chromatogram of 5-formylcytidine monophosphate in digested mitochondrial tRNAs upon loss of SHMT2. The same samples were analysed for 5-taurinomethyluridine monophosphate (p-τm5U) in Fig. 4b. The combined data demonstrate that SHMT2 deletion causes loss of τm5U but not 5-formylcytidine. b, Levels of τm5U, 5-taurinomethyl-2-thiouridine monophosphate (p-τm5s2U) and 2-thiouridine monophosphate (p-s2U) in wild-type HCT116 and SHMT2 deletion lines normalized to 5-formylcytidine monophosphate (p-f5C) (n = 3). c, Taurine levels in HCT116 wild-type and SHMT2-knockout cells (n = 3). d, τm5U levels in digested mitochondrial tRNAs upon re-expression of SHMT2 (n = 1). e, τm5U, τm5s2U and s2U levels normalized to f5C in HCT116 SHMT2/MTHFD2 knockout lines after sarcosine supplementation and HCT116 upon loss of MTO1 (n = 2). For all panels, data are mean ± s.e.m. or individual data points only. f, Labelling pattern of 5-taurinomethyluridine and 5-formylcytidine monophosphate extracted from mitochondrial tRNAs after growth in media containing either [3-13C]serine or [U-13C]methionine. g, Mean cumulative count of ribosome protected fragments (RPF) mapping to mitochondrial protein coding transcripts upon ribosome profiling in HCT116 MTO1-knockout cell lines. Data were normalized to RPM (n = 2); n indicates the number of biological replicates. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values).

Extended Data Figure 8 Investigation of mRNA and protein secondary structure effects on mitochondrial ribosome stalling sites.

a, Identification of mitochondrial RNA secondary structure based on analysis of the mitochondrial transcript data from the dimethyl sulfate sequencing dataset published previously34. R values and Gini differences were calculated to detect changes in nucleotide reactivity between the in vivo and denatured condition for the complete mitochondrial transcriptome. Coloured points indicate structured regions given in Supplementary Table 4. b, Determination of ribosome stalling sites in SHMT2-knockout HCT116 cell lines. Data points represent individual codons of all 13 mitochondrial protein-coding transcripts. For each codon, the y axis indicates the ribosome counts normalized to the gene median in RPM. The x axis indicates the ratio of normalized counts in SHMT2-knockout to normalized counts in wild-type HCT116. Two and three s.d. above the mean of all codons in the genome are indicated by the grey and black dotted line, respectively. Highlighted in red are codons with greater than 2 s.d. c, Mapping of AAG and UUG codons from SHMT2 knockout-specific ribosome stalling sites (>3 s.d.) on protein structures. For b and c, analysis is based on ribosome profiling data in Fig. 3, with two technical replicates of two independent samples. A list of identified codons and mapped AAG and UUG sites is provided in Supplementary Table 5.

Extended Data Figure 9 Mitochondrial transcript codon occupancy from ribosome profiling of individual patient lines.

a, Codon-specific mitochondrial ribosome occupancy ratio (patient/control fibroblasts) in individual patient derived cell lines (n = 1 for each individual patient, normalized to mean of n = 2 control fibroblast lines). Patients either had nuclear MTO1 missense mutations (patient A c.[1261-5T>G];[1430G>A], patient B c.[1222T>A];[1222T>A]) or were diagnosed with MELAS and carry the recurrent point mutation m.3243A>G in the mitochondrial gene for tRNA Leu1 (MT-TL1). b, Next-generation sequencing of mtDNA mutation load m.3243A>G (MT-TL1) in control fibroblasts and MELAS patient cell lines. Each bar shows one biological replicate for control and patient cell lines. Integrative genomics viewer sequencing raw data are shown on the right.

Extended Data Figure 10 Effects of targeting 1C metabolism on mitochondrial function.

a, Mitochondrial complex I and II levels after growth in the absence of folate for five passages or in the presence of the indicated methotrexate concentration for 96 h. Ethidium bromide (250 nM) was used as a positive control. b, Cellular mtDNA levels in HCT116 cells after folate depletion (with or without 100 μM hypoxanthine and 16 μM thymidine (HT) as rescue agents) or in the presence of methotrexate for 96 h (n = 3). c, To determine whether the decrease in respiration due to methotrexate arises from methotrexate depleting mitochondrial DNA, impairing mitochondrial translation, or a combination, in HCT116 cells we compared the effects of methotrexate (50 nM) to ethidium bromide (250 nM = 100 ng ml−1), which is classically used to deplete mitochondrial DNA, and to chloramphenicol (310 μM = 100 μg ml−1), which blocks mitochondrial translation. After 48 h of treatment, methotrexate and ethidium bromide both decreased oxygen consumption and DNA content. Importantly, despite ethidium bromide depleting mitochondrial DNA much more strongly, methotrexate had an equivalent effect on oxygen consumption, consistent with the effect of methotrexate on oxygen consumption being in part via mitochondrial translation inhibition. Data are normalized and compared to untreated control (all n = 3; except oxygen consumption methotrexate 96 h n = 6 and control n = 4). Data are mean ± s.e.m. n indicates the number of biological replicates. *P < 0.01, two-tailed Student’s t-test (see Supplementary Table 7 for exact P values).

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Morscher, R., Ducker, G., Li, S. et al. Mitochondrial translation requires folate-dependent tRNA methylation. Nature 554, 128–132 (2018) doi:10.1038/nature25460

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