Uridine

The CRISPR/Cas9 system was used to create HeLa cell lines lacking the expression of ATP5MG gene encoding ATP synthase subunit g. A pair of guide RNAs targeting exon 1 and exon 2 (see Supplementary Table 1) were subcloned into the BbsI site of px330 plasmid (Addgene). HeLa cells were grown in DMEM (Gibco 11965) supplemented with 10% FBS, 100 mg/L uridine, non-essential amino acids (Gibco), and vitamins (Gibco) in a humidified atmosphere of 5% CO₂/95% air at 37 °C to 70% confluency in 6-well plates. Cells were then transfected with 6 µl Lipofectamine 2000 with 7 µg px330 gRNA1, 7 µg px330 gRNA2, and 7 µg pAAV Syn-GFP (Addgene). The next day, transfected cells were subjected to FAC sorting based on GFP fluorescence and single cells were placed in individual wells of a 96-well plate. The single colonies were subsequently expanded and the loss of subunit g expression was confirmed by Western blot. For cell growth analysis, 10 × 10³ WT or HeLa-Δg cells were seeded into a 6-well plate and counted after 48, 72, and 96 h.

143B‐TK⁻ osteosarcoma‐derived cybrid cells were grown in High‐Glucose (4.5 g/l) DMEM containing Sodium Pyruvate and GlutaMAX™ (Gibco‐Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco‐Thermo Fisher Scientific) and 50 μg/ml uridine (Sigma‐Aldrich). Cells transduced with expression vectors containing a puromycin resistance cassette were grown in the presence of 1 μg/ml puromycin (InvivoGen). If it was hygromycin, the final concentration of the hygromycin B was 100 μg/ml (InvivoGen), and for neomycin‐resistant cells, the final concentration of geneticin was 500 μg/ml (Gibco‐Thermo Fisher Scientific).
The cells used in the SILAC experiments were grown in DMEM for SILAC (Gibco‐Thermo Fisher Scientific) plus 10% dialyzed serum (Gibco‐Thermo Fisher Scientific) and 50 μg/ml uridine, supplemented either with unlabeled L‐lysine monohydrochloride (K₀), L‐arginine (R₀), and L‐proline (“Light” conditions) or with L‐lysine‐¹³C₆,¹⁵N₂ hydrochloride (K₈), L‐arginine‐¹³C₆,¹⁵N₄ hydrochloride (R₁₀), and L‐proline (“Heavy” conditions), all from Sigma‐Aldrich.

The human neuroblastoma cells (SK-N-AS, American Type Culture Collection, Manassas, VA, USA) were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and antibiotic-antimycotic (Invitrogen) in 5% CO₂/95% O₂ humidified incubator at 37 °C. The cells were isolated and then re-cultured at a 1:10 ratio when the cells grew to 60–80% of confluence and maintained in DMEM containing 10% FBS, 25 mM HEPES, 110 mg/mL pyruvate and 50 ng/mL uridine (Invitrogen, Carlsbad, CA, USA) in 5% CO₂/95% O₂ humidified incubator at 37 °C. Furthermore, cells grew in a medium with 50 ng/mL ethidium bromide (EtBr) for 3 months to deplete mtDNA. A single clone was selected by limit dilution. The mtDNA-depleted ρ⁰ cells were confirmed by detection of mtDNA copy number, expression of mtDNA-coded proteins and oxygen consumption in the absence of EtBr [20 (link),21 (link)]. The ρ⁰ cells were subcultured at a 1:3 ratio when the cells grew to 60–80% of confluence.

The Flp-In-TRex human embryonic kidney 293T (HEK293T) cell line (Invitrogen) was cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) tetracycline-free FBS, 2 mM Glutamax (Gibco), 1× penicillin–streptomycin (Gibco), 50 µg ml⁻¹ uridine, 10 µg ml⁻¹ Zeocin (Invitrogen) and 100 µg ml⁻¹ blasticidin (Gibco) at 37 °C under 5% CO₂ atmosphere. Cell lines have been routinely tested for mycoplasma contamination.
For SILAC-based quantitative proteomic analysis, cells were grown in Iscove's modified Dulbecco's medium (IMDM) for Stable Isotope Labeling by Amino acids in Cell culture (SILAC) supplemented with ‘heavy’ (¹⁵N-labelled and ¹³C-labelled) or ‘light’ Arg, Lys and Pro and 10% dialysed FCS (Thermo Scientific HyClone).
The Trmt2b-knockout cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), Glutamax (Gibco) and penicillin–streptomycin (Gibco) at 37 °C, 5% CO₂. Large cell culture volumes were grown in Thomson OptimumGrowth 1.6-l and 5-l flasks in a shaker incubator at 110 rpm. For the cryo-EM sample, approximately 10 bln cells of the Trmt2b-knockout cell line were used.

To generate knockout cell lines stably re-expressing the protein of interest (mS37, MRM3 and GTPBP10), the HEK293T cell line that permits doxycycline-inducible expression of the gene of interest in a dose-dependent manner was used. HEK293T cells were cultured at 37 °C under 5% CO₂ in DMEM supplemented with 10% (v/v) tetracycline-free FBS, 1× penicillin–streptomycin (Gibco), 50 μg ml⁻¹ uridine, 100 μg ml⁻¹ zeocin (Invitrogen) and 10 μg ml⁻¹ blasticidin S (Gibco). Knockout cells were seeded in a 10-cm dish, 1 day before transfection, to achieve 70–90% confluency. Co-transfection of the expression plasmid pcDN5/FRT/TO (with mS37, MRM3-Flag and GTPBP10-Flag) and the Flp-recombinase plasmid pOG44 was carried out using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions. Selective antibiotics, 100 μg ml⁻¹ of hygromycin (Invitrogen) and 10 μg ml⁻¹ blasticidin S were added 48 h post-transfection and culture media were replaced every 2–3 days. To induce expression of the protein of interest, cells were incubated with 50 ng ml⁻¹ doxycycline for 48 h before analysis.

YG (5 g/L yeast extract (Fisher Scientific, Pittsburg, PA, USA), 20 g/L D-glucose (Sigma-Aldrich, St. Louis, MO, USA) and 800 μL of a trace element solution [13 (link)]) was used as a complete medium. For strains carrying pyrG mutations it was supplemented with 10 mM uridine (Fisher Scientific, Pittsburg, PA, USA) and 8.9 mM uracil (Beantown Chemicals, Hudson, NH, USA). Because some batches of yeast extract are deficient in riboflavin, we added riboflavin (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 2.5 μg/mL. Solid complete medium was YAG (YG + 15 g/L agar (Technova, Holister, CA, USA)). Solid minimal medium was 10 g/L D-glucose, 15 g/L agar, 2 mL/L of a 26% w/v MgSO₄∙7H₂O (Avantor, Radnor, PA, USA) solution, 800 μL/L trace element solution [13 (link)], 6 g/L NaNO₃ (Fisher Scientific, Pittsburg, PA, USA), 0.52 g/L KCl (Sigma-Aldrich, St. Louis, MO, USA) and 1.52 g/L KH₂PO₄ (Sigma-Aldrich, St. Louis, MO, USA) (all values are final concentrations). The latter three salts were from a 10X stock salts solution adjusted to pH 6.0–6.5 with saturated KOH (Sigma-Aldrich, St. Louis, MO, USA), autoclaved separately from the other components, cooled to 60 °C and mixed with the solution containing the other components (100 mL + 900 mL) to give the stated final concentrations.