ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

Abstract

ALKBH1 is a 2-oxoglutarate- and Fe²⁺-dependent dioxygenase responsible for multiple cellular functions. Here, we show that ALKBH1 is involved in biogenesis of 5-hydroxymethyl-2΄-O-methylcytidine (hm⁵Cm) and 5-formyl-2΄-O-methylcytidine (f⁵Cm) at the first position (position 34) of anticodon in cytoplasmic tRNA^Leu, as well as f⁵C at the same position in mitochondrial tRNA^Met. Because f⁵C34 of mitochondrial tRNA^Met is essential for translation of AUA, a non-universal codon in mammalian mitochondria, ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f⁵C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. We reconstituted formation of f⁵C34 on mitochondrial tRNA^Metin vitro, and found that ALKBH1 first hydroxylated m⁵C34 to form hm⁵C34, and then oxidized hm⁵C34 to form f⁵C34. Moreover, we found that the frequency of 1-methyladenosine (m¹A) in two mitochondrial tRNAs increased in ALKBH1-knockout cells, indicating that ALKBH1 also has demethylation activity toward m¹A in mt-tRNAs. Based on these results, we conclude that nuclear and mitochondrial ALKBH1 play distinct roles in tRNA modification.

INTRODUCTION

Cellular RNAs contain numerous chemical modifications that are introduced enzymatically after transcription. More than 130 types of modified nucleosides have been reported in various RNA molecules from all domains of life (1), of which about 80% of them were found in tRNAs. tRNA modifications ensure proper tRNA function of tRNAs by stabilizing tertiary structure, and fine-tuning decoding properties for translation (2). A wide variety of RNA modifications are present at the first position (wobble position or position 34) of tRNA anticodons. These modifications play pivotal roles in accurate and efficient translation by modulating codon–anticodon interaction at the ribosomal A-site (3).

5-Formylcytidine (f⁵C) (Figure 1A), first discovered in mammalian mitochondrial (mt) tRNA (Figure 1B) (4), is a unique modification found at position 34 of mt-tRNAs^Met from vertebrates and nematodes (4–6). In mammalian mitochondria, the AUA codon specifies Met instead of Ile (7). In vitro translation revealed that f⁵C34 enables mt-tRNA^Met to recognize AUA as well as AUG (8). Because the sole mt-tRNA^Met acts in both initiation and elongation in mitochondrial protein synthesis, f⁵C34 is an essential modification for mitochondrial protein synthesis. A structural study of the codon–anticodon interaction at the ribosomal A-site revealed that f⁵C34 pairs with adenosine at the third letter of the AUA codon in a canonical Watson–Crick geometry, implying that f⁵C undergoes imino-oxo tautomerization to pair with AUA (9). Since the discovery of this modification, however, biogenesis and physiological role of f⁵C34 remained unresolved. Last year, we demonstrated that biogenesis of f⁵C34 is initiated by S-adenosylmethionine (AdoMet)-dependent methylation catalyzed by NSUN3 (6). Knockout (KO) of NSUN3 resulted in severe reduction in mitochondrial protein synthesis and respiratory activity, leading to mitochondrial dysfunction. We successfully reconstituted formation of 5-methylcytidine (m⁵C) at the wobble position of mt-tRNA^Met with recombinant NSUN3 in the presence of AdoMet, suggesting that f⁵C34 is synthesized by NSUN3-mediated m⁵C34 formation, followed by hydroxylation and oxidation of the methyl group.

Figure 1.

5-Formylcytidine (f⁵C) and derivatives found in human tRNAs. (A) Chemical structures of 5-formylcytidine (f⁵C), 5- hydroxymethyl-2΄-O-methylcytidine (hm⁵Cm) and 5-formyl-2΄-O-methylcytidine (f⁵Cm). Modified groups are shown in red. (B) Secondary structures of human ct-tRNA^Leu(CAA) (left) and mt-tRNA^Met (right) with post-transcriptional modifications: 2-methylguanosine (m²G), N⁴-acetylcytidine (ac⁴C), dihydrouridine (D), pseudouridine (Ψ), 2,2-dimethylguanosine (m^2,2G), 1-methylguanosine (m¹G), 2΄-O-methyluridine (Um), 5-methylcytidine (m⁵C), 5-methyluridine (m⁵U) and 1-methyladenosine (m¹A). f⁵C and derivatives are shown in red. Modified positions are numbered as previously described (41,67). Eight pathogenic point mutations are indicated in human mt-tRNA^Met.

We identified two pathogenic point mutations (A4435G and C4437U) in mt-tRNA^Met (Figure 1B) that impaired m⁵C34 formation by NSUN3 (6). These mutations are associated with mitochondrial diseases presenting with maternally inherited hypertension, Leber's hereditary optic neuropathy, hypotonia, seizure, muscle weakness, lactic acidosis and hearing loss (10–12). In addition, a loss-of-function mutation in NSUN3 was identified in a patient presenting with symptoms of mitochondrial disorder, including developmental disability, microcephaly, muscular weakness, external ophthalmoplegia and lactic acidosis (13,14). These observations indicated that loss of f⁵C34 or NSUN3 results in pathological consequence.

In a previous study, ALKBH1 could oxidize m⁵C34 in the anticodon-stem loop of mt-tRNA^Met to form f⁵C34 in vitro (15), although hm⁵C34 was not detected. Indirect detection of f⁵C34 using chemical derivatization combined with primer extension revealed that the fraction of f⁵C34-containing mt-tRNA^Met decreased upon ALKBH1 knockdown, indicating that ALKBH1 is involved in f⁵C34 formation. However, hm⁵C34 and m⁵C34 could not be distinguished in this experiment, and it remained unclear to what extent ALKBH1 contributes to this process in the cell. Considering that ALKBH1 is involved in demethylation of N⁶-methyldeoxyadenosine (m⁶dA) in genomic DNA (16) and demethylation of 1-methyladenosine (m¹A) in cytoplasmic (ct-)tRNAs (17), it was necessary to perform a more sophisticated analysis of the modification status of tRNAs with the goal of obtaining direct evidence for ALKBH1 as an RNA oxygenase responsible for f⁵C34 formation.

On the other hand, 2΄-O-methyl derivative of f⁵C (5-formyl-2΄-O-methylcytidine, f⁵Cm) (Figure 1A) is present at the wobble position of mammalian ct-tRNA^Leu with the CAA anticodon (Figure 1B) (18). Given that f⁵C allows tRNA to pair with A (8), this modification has been speculated to permit the recognition of UUA in addition to UUG. Notably, the 5-hydroxymethylcytidine derivative hm⁵Cm (Figure 1A), a reduced form of f⁵Cm, has also been detected in this tRNA (18), strongly indicating that f⁵Cm and hm⁵Cm are synthesized via oxidation of m⁵C. A metabolic labeling study revealed that hm⁵C and f⁵C in mouse total RNA are generated by oxidation of m⁵C (19), indicating that hm⁵Cm and f⁵Cm in ct-tRNA^Leu(CAA) are synthesized by hydroxylation and oxidation of m⁵C. However, the RNA dioxygenase responsible for this process has not been previously identified.

Here, we report that ALKBH1 is an RNA dioxygenase involved in biogenesis of hm⁵Cm34 and f⁵Cm34 in ct-tRNA^Leu, as well as f⁵C34 in mt-tRNA^Met, ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced oxygen consumption, indicating that ALKBH1 is required for efficient mitochondrial activity. We successfully reconstituted formation of hm⁵C and f⁵C with ALKBH1 in the presence of 2-oxoglutarate (2-OG) and Fe²⁺, demonstrating that f⁵C is synthesized by consecutive hydroxylation and oxidation of m⁵C. We also examined several pathogenic mutations in mt-tRNA^Met associated with mitochondrial diseases, but these mutations had little effect on f⁵C formation mediated by ALKBH1, indicating that these diseases are caused by lack of f⁵C in the mutant tRNAs resulting from defective methylation by NSUN3, but not from defective oxidation by ALKBH1. Moreover, we found that the frequency of m¹A in two mt-tRNAs increased in ALKBH1 KO cells, indicating that ALKBH1 also has demethylation activity toward m¹A in some mt-tRNAs.

MATERIALS AND METHODS

Cell culture and measurement of cell proliferation

HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C under a humidified atmosphere containing 5% CO₂. ALKBH1 KO and NSUN3 KO HEK293T cells were cultured in DMEM (high-glucose) supplemented with 10% FBS, 1% penicillin–streptomycin, 0.11 g/l sodium pyruvate and 0.05 g/l uridine. For measuring cell proliferation, wild-type (WT) and KO HEK293T cells were seeded on 96-well plates (8.0 × 10³ cells per well) in glucose medium (no-glucose DMEM supplemented with 2 mg/ml glucose) or galactose medium (no-glucose DMEM supplemented with 2 mg/ml galactose) supplemented with alamarBlue reagent (Invitrogen). The plates were incubated at 37°C for 3 h, and the absorbances at 570 and 600 nm were measured on a SpectraMax 190 microplate reader (Molecular Devices). Cell proliferation was evaluated based on the percentage of reduced dye.

Construction of ALKBH1 KO lines

The human ALKBH1 gene was knocked out using the CRISPR-Cas9 system basically as described (20). We designed an sgRNA targeting exon 4 of ALKBH1 gene. Sense and antisense DNAs for the sgRNA (Supplementary Table S1) were cloned into vector pX330 (Addgene plasmid 42230). HEK293T cells (2.0 × 10⁵ cells per well) were transfected with the cloned vector (300 ng) and seeded on 24-well plates. The KO efficiency of each sgRNA was evaluated by surveyor assay using a specific pair of primers (Supplementary Table S1). The cells were diluted and sub-cultured on 10 cm dishes. Eleven days after the transfection, colonies were isolated and genomic DNAs were extracted. ALKBH1 KO lines were selected by confirming the frameshift mutations in the target region.

RNA extraction and tRNA isolation

Total RNA was extracted from HEK293T cells using TriPure Isolation Reagent (Roche Life Science). Total RNA from mouse fetus was kindly provided by S. Nakagawa (Hokkaido University). Individual tRNAs were isolated by reciprocal circulating chromatography (RCC) (21,22) using the 5΄-terminal ethylcarbamate amino-modified DNA probes listed in Supplementary Table S1.

RNA mass spectrometry

Isolated tRNA (1–2 pmol) was digested with RNase T₁, RNase A or MazF (Takara Bio.) at 37°C for 30 min in 20 mM NH₄OAc (pH 5.3), followed by mixing with an equal volume of 100 mM triethylamine–acetate and subjected to capillary liquid chromatography (LC)–nano–electrospray ionization (ESI)–mass spectrometry (MS) as described (22–24).

cDNA cloning and expression of recombinant ALKBH1

The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers (Supplementary Table S1) and cloned into pE-SUMO-NSUN3 (6) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1. Escherichia coli Rosetta2 (DE3) (Novagen) was transformed with pE-SUMO-ALKBH1 and cultured at 25°C. When the OD₆₀₀ reached 0.4, 10 μM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the culture was incubated at 16°C for an additional 24 h. The harvested cells were lysed by sonication in a buffer consisting of 20 mM Tris–HCl (pH 7.5), 0.5 M KCl, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethanesulfonyl fluoride (PMSF). Soluble protein was loaded onto a HisTrap HP column (GE Healthcare) on an AKTA Purifier 10 system (GE Healthcare) and separated with 0–500 mM gradient of imidazole. Fractions containing His6-SUMO-tagged human ALKBH1 were collected and dialyzed overnight in buffer consisting of 20 mM Tris–HCl (pH 7.5), 0.5 M KCl, 1 mM DTT and 10% glycerol. The His6-SUMO tag was cleaved by ubiquitin-like specific protease 1 (Ulp1) at 4°C overnight, and then removed by passing the sample through HisLink protein proliferation resin (Promega). The untagged protein was equilibrated with a buffer consisting of 50 mM Tris–HCl (pH 8.0), 50 mM NaCl and 7 mM 2-mercaptoethanol, and further purified by Mono Q anion exchange chromatography (HR 5/5, GE Healthcare) with a gradient of NaCl (50–1000 mM) using AKTA Purifier 10 system (GE Healthcare). The purified ALKBH1 was dialyzed against a buffer consisting of 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 7 mM 2-mercaptoethanol, followed by addition of glycerol to 30% f.c.

In vitro reconstitution of f⁵C34 by recombinant ALKBH1

WT and mutant mt-tRNAs^Met were prepared by in vitro transcription using T7 RNA polymerase, basically as described (25). Synthetic DNAs for templates are listed in Supplementary Table S1. Anticodon stem-loop with m⁵C34 was chemically synthesized by GeneDesign Inc. The in vitro transcribed mt-tRNA^Met was methylated by recombinant NSUN3 (6) at 37°C for 90 min in a reaction mixture consisting of 20 mM HEPES–KOH (pH 8.0), 5 mM MgCl₂, 100 mM KCl, 1 mM DTT, 2.8 μM mt-tRNA^Met transcript, 2.8 μM NSUN3 and 640 μM AdoMet. tRNA containing m⁵C34 was extracted with phenol:chloroform:isoamylalcohol (25:24:1, v/v/v) and collected by ethanol precipitation. In vitro reconstitution of f⁵C34 was performed at 37°C for 90 min in a reaction mixture containing 20 mM HEPES–KOH (pH 6.8), 5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 4 mM ascorbic acid, 100 μM 2-OG, 80 μM (NH₄)₂Fe(SO₄)₂·6H₂O, 0.5 μM mt-tRNA^Met with m⁵C34 and 0.125 μM recombinant ALKBH1. For the series of mt-tRNA^Met mutants, incubation time was 15 min. The reaction was stopped by addition of an equal volume of phenol:chloroform:isoamylalcohol (25:24:1, v/v/v), and tRNAs were precipitated with ethanol. The tRNA was digested with RNase T₁ and subjected to LC/MS to analyze modification status. For in vitro reconstitution of f⁵C34 from C34, the unmodified mt-tRNA^Met was incubated at 37°C in a reaction mixture consisting of 20 mM HEPES–KOH (pH 8.0), 5 mM MgCl₂, 50 mM KCl, 1 mM DTT, 230 μM AdoMet, 4 mM ascorbic acid, 100 μM 2-OG, 80 μM (NH₄)₂Fe(SO₄)₂·6H₂O, 0.5 μM tRNA transcript, 0.5 μM NSUN3 and 0.125 μM ALKBH1. Aliquots were taken at the indicated times (Figure 6C) and subjected to LC/MS as described above.

Figure 6.

In vitro reconstitution of f⁵C34 formation (A) Mass chromatograms of the anticodon-containing fragments of mt-tRNA^Met and ASL to detect f⁵C34 (top), hm⁵C34 (middle) and m⁵C34 (bottom) after in vitro reconstitution of f⁵C34 with ALKBH1 in the presence or absence of 2-OG and Fe²⁺. (B) CID spectra of the hm⁵C34 (left)- and f⁵C34 (right)-containing fragments reconstituted in vitro. The precursor ions are m/z 1163.48 and 1163.15 for hm⁵C34 and f⁵C34, respectively. Product ions are assigned on the corresponding sequences. (C) In vitro f⁵C34 formation from C34 via consecutive reactions mediated by NSUN3 and ALKBH1. Time-dependent alteration of modification status in mt-tRNA^Met. Fractions of the RNase T₁-digested fragments bearing C34 (green), m⁵C34 (pale green), hm⁵C34 (pale orange) and f⁵C34 (orange) at each time point were calculated based on peak heights of mass chromatograms. Because hm⁵C has a molecular mass 2Da larger than that of f⁵C, the frequency of hm⁵C was calculated by subtracting the theoretical value of the second isotopic peak (+2) of the f⁵C-containing fragment. (D) Methylation (gray bars) and oxidation (orange bars) efficiencies of mutant mt-tRNAs^Met normalized against those of mt-tRNA^Met. Positions of pathogenic mutations in mt-tRNA^Met are shown in Figure 1B. Methylation data were obtained from our previous study (6). Each mt-tRNA^Met mutant containing m⁵C34 was incubated with ALKBH1 in the presence of 2-OG and Fe²⁺, followed by RNase T₁ digestion and subjected to RNA-MS. Reaction efficiency was calculated by summing the peak heights of the anticodon-containing fragments bearing hm⁵C34 and f⁵C34, normalized against the total peak heights of the three fragments (m⁵C34, hm⁵C34 and f⁵C34). Frequency of hm⁵C was calculated by subtracting the theoretical value of the second isotopic peak (+2) of the f⁵C-containing fragment. Data are presented as means ± SD of three independent reactions. *P < 0.001, Student's t-test.

Mitochondrial activities

HEK293T WT, ALKBH1 KO and NSUN3 KO cells (4.0 × 10⁷) were suspended in 3 ml MOPS buffer consisting of 10 mM MOPS, 250 mM sucrose and 1 × Complete Mini Protease Inhibitor Cocktail Tablet (Roche), and then homogenized on ice by 12 up-and-down strokes with a Teflon homogenizer. The homogenate was centrifuged at 600 × g for 5 min at 4°C, and the supernatant was further centrifuged at 10 000 × g for 15 min at 4°C. The mitochondrial pellet was collected and resuspended in MOPS buffer. The activities of respiratory chain complexes were evaluated as described (26). The oxygen consumption rate (OCR) of WT, ALKBH1 KO and NSUN3 KO cells was measured using an XF24-3 extracellular flux analyzer (Seahorse Bioscience).

Pulse labeling of mitochondrial protein synthesis

HEK293T WT, ALKBH1 KO and NSUN3 KO cells (2.0 × 10⁴) were seeded on 10 cm dishes and cultured overnight. Cells were washed with L-Met-, L-Gln- and L-Cys-free medium (Sigma-Aldrich) supplemented with 2 mM L-Gln, 10% FBS and 1 mM sodium pyruvate. To inhibit cytoplasmic protein synthesis, 50 μg/ml emetine was added to the medium and incubated for 10 min. The cells were further supplemented with 8.14 MBq (0.22 mCi) of [³⁵S] Met/[³⁵S] Cys (EXPRE³⁵S³⁵S Protein Labeling Mix, [³⁵S]-, PerkinElmer) and incubated for 1 h to label proteins translated in mitochondria. Then, the medium was replaced with cold medium (DMEM with 10% FBS) and chased for 10 min, and the cells were harvested by centrifugation at 1500 × g for 10 min at 4°C. The cell lysates were resolved by Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (16.5%), and the gel was coomassie brilliant blue (CBB)-stained and dried by a gel drier (AE-3750 RapiDry, ATTO). The dried gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) and visualized on a FLA-7000 fluorimager (Fujifilm).

Primer extension to detect m¹A modification

Primer extension was conducted basically as described (27,28). The 5΄ ³²P-labeled primer (0.1 pmol) was incubated with 3.125 μg total RNA in a 5 μl mixture containing 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 2 min at 80°C, followed by cooling to room temperature. Subsequently, 0.5 μl (100 units) SuperScript III (Invitrogen) was added to 4.5 μl of the premixed solution [2 μl 5 × FS buffer (Invitrogen), 0.25 μl of 1.5 mM d/ddNTP mix, 0.75 μl ddH₂O and 1.5 μl of 25 mM MgCl₂]. The d/ddNTP mix consisted of dTTP, dGTP and ddCTP for mt-tRNA^Lys; dTTP, dGTP and ddATP for mt-tRNA^Arg; and dATP, dTTP, dCTP and ddGTP for mitochondrial 16S rRNA. The reaction was carried out for 1 h at 47°C for mt-tRNAs and 55°C for mitochondrial 16S rRNA. The reaction was stopped by addition of 0.5 μl of 4 M NaOH and boiling for 5 min at 95°C, and then neutralized by addition of 4.5 μl of 1 M Tris–HCl (pH 5.0). The cDNAs were resolved by 20% denaturing PAGE. The gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) and visualized on a FLA-7000 fluorimager (Fujifilm).

RESULTS

Modification status of precursors and mature ct-tRNA^Leu (CAA)

In Saccharomyces cerevisiae and vertebrates, ct-tRNA^Leu(CAA) has an intron in its anticodon region (29,30), indicating that biogenesis of f⁵Cm34 is coupled with intron removal via tRNA splicing. To study the biogenesis of f⁵Cm34 in ct-tRNA^Leu(CAA), we first analyzed the modification status of their precursors by mass-spectrometric analysis (RNA-MS) and observed sequential formation of f⁵Cm34. From mouse fetus, we isolated two precursors of ct-tRNA^Leu(CAA), along with its mature form by RCC (Figure 2A) (21). The isolated tRNAs were digested by RNase T₁ and subjected to RNA-MS to detect the anticodon-containing fragments (Figure 2B), which were then further probed by collision-induced dissociation (CID) to map the modified residues (Supplementary Figure S1). The primary transcript of ct-tRNA^Leu(CAA), bearing the 5΄ leader, 3΄ trailer and intron, mainly contained m⁵C34. Only 1.2% of this precursor contained hm⁵C34, and no f⁵C34 was detected. On the other hand, the post-spliced precursor contained m⁵C34 and hm⁵C34 as major modifications, and f⁵C34 was present in 6.9% of this precursor. In the mature ct-tRNA^Leu(CAA), hm⁵C34m and f⁵Cm34 were abundant. These results revealed the order and timing of each step of f⁵Cm34 formation. Given that Trm4/NSUN2 methylates C34 in ct-tRNA^Leu(CAA) in an intron-dependent manner (31,32), m⁵C34 must be introduced by NSUN2 in the primary transcript in the nucleus. After removal of the intron from the primary transcript, the post-spliced tRNA precursor undergoes hydroxylation and oxidation by an unknown enzyme to yield hm⁵C34 and f⁵C34. Followed by 5΄ and 3΄ processing and CCA addition, tRNA precursor is exported to the cytoplasm, where 2΄-O-methylation takes place to yield hm⁵Cm34 and f⁵Cm34.

Figure 2.

Modification status at the wobble position of precursor and mature ct-tRNA^Leu (CAA) (A) Isolation of ct-tRNA^Leu(CAA) from mouse fetus. Total RNA and isolated ct-tRNA^Leu(CAA) were resolved by electrophoresis on 10% polyacrylamide gels containing 7 M urea and stained with SYBR Gold. RNA bands for primary, spliced and mature tRNAs are indicated on the right. (B) Mass chromatograms of anticodon-containing fragments with f⁵Cm34 (top), hm⁵Cm34 (second), f⁵C34 (third), hm⁵C34 (forth), m⁵C34 (fifth) and C34 (bottom) from primary (left), spliced (center) and mature (right) forms of ct-tRNA^Leu(CAA). The sequence of each fragment with m/z value and charge state is indicated. Frequency (%) of fragment with each modification is indicated. n.d., not detected. Gray triangles indicate target peaks, while asterisk indicates an unidentified fragment whose molecular mass differs from that of the target fragment. The 7-mer-fragments (positions 31−37) were detected in the primary tRNA, as G37 remained unmodified, whereas the 12-mer-fragments (positions 31−42) were detected due to the presence of m¹G37 in the spliced and mature tRNAs.

Biogenesis of hm⁵Cm34 and f⁵Cm34 in ct-tRNA^Leu(CAA)

According to our analyses of ct-tRNA^Leu(CAA), hydroxylation and oxidation of hm⁵Cm34/f⁵Cm34 must occur in the nucleus because the post-spliced precursor that had not yet undergone end-maturation already contained hm⁵C34 and f⁵C34 (Figure 2B). We chose ALKBH1 as a candidate for the RNA dioxygenase responsible for this process based on its previously reported nuclear functions (16,33). We knocked out this gene in HEK293T cells using the CRISPR-Cas9 system and obtained two KO cell lines in which both alleles contained frameshift mutations (Figure 3A). Next, we isolated ct-tRNA^Leu(CAA) from ALKBH1 KO cells and subjected it to RNA-MS to analyze its modification status (Figure 3B). In WT HEK293T cells, f⁵Cm34 and hm⁵Cm34 were abundantly detected in ct-tRNA^Leu(CAA). In ALKBH1 KO cells, neither f⁵Cm34 nor hm⁵Cm34 were present, and instead m⁵Cm34 was found, demonstrating that ALKBH1 is responsible for conversion from m⁵Cm34 to f⁵Cm34/hm⁵Cm34. Next, we selected FTSJ1 as a strong candidate for the tRNA methyltransferase responsible for 2΄-O-methylation of f⁵Cm34/hm⁵Cm34, because FTSJ1 is localized in the cytoplasm and is involved in 2΄-O-methylation of residues at positions 32 and 34 in the anticodon region (34,35). We knocked out this gene and obtained a FTSJ1 KO cell line (Figure 3C). Ct-tRNA^Leu(CAA) was isolated from the KO cells and subjected to RNA-MS. As expected, both the f⁵Cm34 and hm⁵Cm34 detected in WT cells disappeared, and f⁵C34 and hm⁵C34 increased, in FTSJ1 KO cells (Figure 3D), demonstrating that FTSJ1 is responsible for 2΄-O-methylation of f⁵Cm34/hm⁵Cm34. According to yeast studies (36), Trm7p, an S. cerevisiae homolog of FTSJ1, introduces 2΄-O-methylation at positions 32 and 34. To select different positions for methylation, FTSJ1 chooses two kinds of partner proteins, Trm732p and Trm734p, for positions 32 and 34, respectively. In human, as WDR6 is an apparent homolog of Trm734p, FTSJ1 complexed with WDR6 is likely to be involved in this process (Figure 3E).

Figure 3.

Genetic analyses of hm⁵Cm34/f⁵Cm34 formation in ct-tRNA^Leu(CAA) (A) Schematic depiction of the human ALKBH1 gene and target site of mutations introduced by the CRISPR–Cas9 system. Shaded and open boxes indicate coding regions and untranslated regions of exons, respectively. Lines indicate introns. The target sequence of sgRNA in exon 4 is underlined, and the proto-spacer adjacent motif (PAM) sequence is boxed. Sequences of both alleles in KO1 and KO2 cell lines are aligned. Deleted nucleotides are indicated as dashed lines, and the inserted nucleotide is indicated by a gray letter. (B) Mass chromatograms of the anticodon-containing fragments of ct-tRNA^Leu(CAA) containing f⁵Cm34 (top), hm⁵Cm34 (middle) and m⁵Cm34 (bottom) isolated from WT (left) and ALKBH1 KO (right) cell lines. n.d., not detected. Target peaks are indicated by gray triangles. Filled diamond in the left middle panel indicates a trace of an isotopic ion of f⁵Cm-containing fragment. Asterisk indicates an unidentified fragment whose molecular mass differs from that of the target fragment. (C) Schematic depiction of the human FTSJ1 gene and target site of the mutation introduced by the CRISPR–Cas9 system. Shaded and open boxes indicate coding regions and untranslated regions of exons, respectively. Lines indicate introns. The target sequence of sgRNA in exon 2 is underlined, and the PAM sequence is boxed. Sequences of both alleles in KO1 cell lines are aligned. Deleted nucleotides are indicated by dashed lines. (D) Mass chromatograms of the anticodon-containing fragments of ct-tRNA^Leu(CAA) having f⁵Cm34 (top), hm⁵Cm34 (second), f⁵C34 (third) and hm⁵C34 (bottom) isolated from WT (left) and FTSJ1 KO (right) cell lines. n.d., not detected. Target peaks are indicated by gray triangles. Filled diamond in the left second panel indicates a trace isotopic ion of the f⁵Cm-containing fragment. Asterisk indicates an unidentified fragment whose molecular mass differs from that of the target fragment. (E) Biosynthetic pathways of hm⁵Cm34 and f⁵Cm34 in ct-tRNA^Leu(CAA). Initially, NSUN2 methylates C34 to form m⁵C34 in the presence of AdoMet. ALKBH1 hydroxylates m⁵C34 to form hm⁵C34, and then oxidizes hm⁵C34 to yield f⁵C34. These reactions require O₂ and 2-OG as substrates. Precursor ct-tRNA^Leu(CAA) bearing hm⁵C34 or f⁵C34 is exported to the cytoplasm and methylated by FTSJ1 together with a partner protein (probably WDR6) in an AdoMet-dependent manner to form hm⁵Cm34 or f⁵Cm34, respectively. In ALKBH1 KO cells (shaded), ct-tRNA^Leu(CAA) containing m⁵C34 is exported to the cytoplasm, where it is methylated by FTSJ1 to form m⁵Cm34.

Taken together, these results elucidate the biogenesis of hm⁵Cm34 and f⁵Cm34 in ct-tRNA^Leu(CAA) (Figure 3E). First, NSUN2 methylates C34 to give m⁵C34 in the primary transcript of ct-tRNA^Leu(CAA) in the nucleus (32), followed by tRNA splicing to remove the intron in the anticodon loop. Then, m⁵C34 in the post-spliced tRNA precursor is partially hydroxylated to form hm⁵C34 and further oxidized to form f⁵C34. During this process, the 5΄ leader and 3΄ trailer of the precursor are removed to become an end-matured precursor, which is then exported to the cytoplasm, where the wobble bases are 2΄-O-methylated by FTSJ1 together with a partner protein (probably WDR6) to form hm⁵Cm34 and f⁵Cm34. On the other hand, in ALKBH1 KO cells, ct-tRNA^Leu(CAA) containing m⁵C34 is exported to the cytoplasm and methylated by FTSJ1 to form m⁵Cm34.

Biogenesis of f⁵C34 in mt-tRNA^Met

ALKBH1 is also localized in mitochondria (37). Hence, we investigated whether ALKBH1 is involved in f⁵C formation of mt-tRNA^Met. For this purpose, we isolated mt-tRNA^Met from WT HEK293T cells and the ALKBH1 KO cells, and analyzed the wobble modification by RNA-MS. As shown in Figure 4A, f⁵C34 was fully introduced in mt-tRNA^Met isolated from WT cells, whereas no hm⁵C34, m⁵C34 or C34 was clearly present. On the other hand, f⁵C34 was completely absent, whereas m⁵C34 was clearly present, in mt-tRNA^Met isolated from ALKBH1 KO cells, demonstrating that ALKBH1 is responsible for conversion from m⁵C34 to f⁵C34 in mt-tRNA^Met, and that no other enzymes are redundantly involved in this process. To confirm this result, we introduced plasmid-encoded ALKBH1 into KO cells to rescue f⁵C34 formation. RNA-MS analysis of the isolated mt-tRNA^Met revealed that f⁵C34 was restored (Figure 4A), albeit partially, probably due to inefficient transfection.

Figure 4.

ALKBH1 is essential for f⁵C34 biogenesis in mt-tRNA^Met. (A) Mass chromatograms of the anticodon-containing fragments of mt-tRNA^Met containing f⁵C34 (top), hm⁵C34 (second), m⁵C34 (third) and C34 (bottom) isolated from WT, ALKBH1 KO cell lines and KO1 rescued with plasmid-encoded ALKBH1. n.d., not detected. Filled diamonds in the second panels indicate a trace isotopic ion of the f⁵C-containing fragment. (B) Biosynthetic pathway of f⁵C34 in mt-tRNA^Met. NSUN3 methylates C34 to form m⁵C34 in the presence of AdoMet. ALKBH1 hydroxylates m⁵C34 to form hm⁵C34, and then oxidizes hm⁵C34 to yield f⁵C34. These reactions require O₂ and 2-OG as substrates.

Based on these findings, we propose a reaction scheme for biogenesis of f⁵C34 in mt-tRNA^Met (Figure 4B). In this pathway, NSUN3 initiates formation of f⁵C34 in mt-tRNA^Met by AdoMet-dependent methylation of C34 to form m⁵C34. Then, ALKBH1 hydroxylates m⁵C34 to form hm⁵C34, followed by oxidation of hm⁵C34 to yield f⁵C34.

Deletion of ALKBH1 causes mitochondrial dysfunction

Because loss of f⁵C in NSUN3 KO cells results in mitochondrial dysfunction (6), we next examined the growth properties of ALKBH1 KO cells in glucose and galactose media (Figure 5A). Mitochondrial activity is required for growth in a medium containing galactose as the primary carbon source (38). ALKBH1 KO cells grew as well as NSUN3 KO cells in glucose medium, but a bit slower than WT cells. Compared to WT cells, ALKBH1 KO cells grew much more slowly in galactose medium, as also observed for NSUN3 KO cells, indicating that ALKBH1 is required for efficient mitochondrial activity. Next, we biochemically compared respiratory complex activities between ALKBH1 KO, NSUN3 KO and WT cells (Figure 5B). We observed a clear reduction of Complex I activity in ALKBH1 KO and NSUN3 KO cells, but no significant changes in the other complexes. The OCRs of ALKBH1 KO and NSUN3 KO cells were substantially lower than that of WT cells (Figure 5C), indicating that oxidative phosphorylation and respiratory activity of mitochondria were impaired by ALKBH1 KO.

Figure 5.

ALKBH1 is required for mitochondrial function. (A) Growth curves of WT, ALKBH1 KO and NSUN3 KO cells in a medium containing glucose (left panel) or galactose (right panel) as a primary carbon source. Mean values of five independent cultures are plotted; error bars indicate s.d. (B) Relative activities of respiratory chain complexes in WT, ALKBH1 KO and NSUN3 KO cells. Activity of each complex is normalized against the corresponding citrate synthase (CS) activity. Data are presented as mean ± SD. of three independent assays. *P < 0.05, Student's t-test. (C) Oxygen consumption rate of WT, ALKBH1 KO and NSUN3 KO cells, measured by an XF24 extracellular flux analyzer. Data are presented as mean ± SD. of three independent assays. *P < 0.005, Student's t-test. (D) Pulse labeling of mitochondrial protein synthesis in WT, ALKBH1 KO and NSUN3 KO cells. Under inhibition of cytoplasmic translation, mitochondrial protein synthesis was labeled with [³⁵S] Met and [³⁵S] Cys.

To examine mitochondrial protein synthesis, we conducted a pulse-labeling experiment in ALKBH1 KO and NSUN3 KO cells (Figure 5D). In cells treated with emetine to inhibit cytoplasmic translation, mitochondrial translation was specifically labeled by addition of [³⁵S] Met and [³⁵S] Cys for short periods of time. We found that mitochondrial protein synthesis was substantially reduced in ALKBH1 KO and NSUN3 KO cells in comparison to WT cells. This finding suggests that m⁵C34 in mt-tRNA^Met in ALKBH1 KO cells cannot efficiently decipher the AUA codon.

In vitro reconstitution of f⁵C modification

To examine the enzymatic activity of ALKBH1 responsible for f⁵C formation, we performed in vitro reconstitution of f⁵C34 in mt-tRNA^Met. For this purpose, in vitro transcribed mt-tRNA^Met containing m⁵C34 introduced by NSUN3 was incubated with recombinant ALKBH1 in the presence or absence of 2-OG and Fe²⁺. The products were digested with RNase T₁ and subjected to RNA-MS (Figure 6A). We detected an f⁵C34-containing fragment, along with a hm⁵C34-containing fragment, only in the presence of both 2-OG and Fe²⁺. These modified fragments were further probed by CID to confirm the presence of hm⁵C and f⁵C at position 34 (Figure 6B). In addition, we used the m⁵C34-containing anticodon stem-loop (ASL) as a substrate (Figure 6A) and found that f⁵C34 and hm⁵C34 were clearly formed in this substrate. We also performed in vitro formation of f⁵C34 from C34 via consecutive reactions mediated by NSUN3 and ALKBH1 (Figure 6C). In this experiment, unmodified mt-tRNA^Met was incubated with NSUN3 and ALKBH1 in the presence of AdoMet, 2-OG and Fe²⁺. During accumulation of m⁵C34, hm⁵C34 and f⁵C34 gradually formed. Ultimately, 95% of tRNAs were methylated by NSUN3 after 2 h. Among the products, 74% contained hm⁵C34, while 23% had f⁵C34. This finding demonstrated that f⁵C34 can be reconstituted by cooperative modifications with NSUN3 and ALKBH1.

These results suggest that ALKBH1 is a 2-OG- and Fe²⁺-dependent RNA dioxygenase that hydroxylates and oxidizes m⁵C34 to form hm⁵C34 and f⁵C34, respectively. Because ASL could be a substrate for f⁵C34/hm⁵C34 formation, ALKBH1 recognizes the anticodon–stem–loop region and does not require the entire tRNA structure for this reaction.

Impact of pathogenic point mutations on f⁵C formation

According to MITOMAP, eight point mutations in the mt-tRNA^Met gene are associated with human diseases (39). We previously reported two pathogenic mutations, A4435G and C4437U, that impaired m⁵C34 formation by NSUN3, possibly causing hypomodification of f⁵C34 (6). To determine whether these point mutations affect f⁵C34 formation mediated by ALKBH1, mutant mt-tRNAs^Met containing each of eight pathogenic mutations were prepared and methylated by NSUN3 to introduce as much m⁵C34 as possible. Then, we examined in vitro hydroxylation and oxidation of m⁵C34 in these mutant tRNAs by ALKBH1, but observed no obvious reduction of hm⁵C34/f⁵C34 formation (Figure 6D). This result suggests that none of the pathogenic point mutations in mt-tRNA^Met tested here impair hydroxylation or oxidation of m⁵C34 mediated by ALKBH1.

ALKBH1 is involved in demethylation of m¹A in mt-tRNAs

ALKBH1 can demethylate m¹A in ct-tRNAs (17). Given that ALKBH1 is localized in mitochondria (37), we asked whether mitochondrial ALKBH1 is also involved in demethylation of m¹A in mitochondrial RNAs. m¹A is present at positions 9, 16 and 58 of several tRNAs (7,40,41) and at position 947 of 16S rRNA (28). We isolated mt-tRNAs for Arg, Lys and Leu(UUR) from WT and ALKBH1 KO cells, and compared the m¹A frequencies among them by measuring m¹A-containing fragments by RNA-MS (Figure 7AB and Supplementary Figure S2A–C). The frequency of m¹A16 in mt-tRNA^Arg was higher in ALKBH1 KO cells (33.3%), than in WT cells (17.9%) (Figure 7A). The level of m¹A58 in mt-tRNA^Lys was also elevated in ALKBH1 KO cells (Figure 7B). The increase in m¹A frequency at these sites in ALKBH1 KO cells was also confirmed by primer extension (Figure 7CD). Other m¹A sites at positions 9 and 58 of mt-tRNAs were unchanged upon KO of ALKBH1 (Supplementary Figure S2A–C). In particular, m¹A58 was fully introduced in mt-tRNA^Leu(UAA), ct-tRNA^Leu(CAA), ct-tRNA^Val(AAC) and ct-tRNA^Asn(GUU) even in WT cells (Supplementary Figure S2C–F). Thus, ALKBH1 is not involved in demethylation of m¹A58 in these tRNAs. We also compared m¹A frequency at position 947 in 16S rRNA by primer extension analysis and found no significant change in the m¹A947 level between WT and ALKBH1 KO cells (Supplementary Figure S2G).

Figure 7.

Level of m¹A in mt-tRNAs increased in ALKBH1 KO cells (A) Mass chromatograms detecting negative ions (z = −4 to −6) of RNase T₁–digested fragments containing A16 (upper) and m¹A16 (lower) of mt-tRNA^Arg isolated from WT and ALKBH1 KO cells. (B) Mass chromatograms detecting negative ions (z = −6 to −8) of RNase T₁–digested fragments containing A58 (upper) and m¹A58 (lower) of mt-tRNA^Lys isolated from WT and ALKBH1 KO cells. (C) Detection of m¹A16 in mt-tRNA^Arg from WT and ALKBH1 KO cells by primer extension. The primer is shown as a black line, and the extended cDNAs for detection of m¹A16 and A16 are depicted as red and blue lines, respectively. (D) Detection of m¹A58 in mt-tRNA^Lys from WT and ALKBH1 KO cells by primer extension. The primer is shown as a black line, and the extended cDNAs for detection of m¹A58 and A58 are depicted as red and blue lines, respectively.

DISCUSSION

Bacterial AlkB is a 2-OG- and Fe²⁺-dependent dioxygenase that repairs alkylation damage, including m¹A and m³C in DNA and RNA (42,43). In mammals, nine members of AlkB-family proteins (ALKBH1-8 and fat mass and obesity-associated protein (FTO)) have been identified (44–47), of which four are associated with RNA metabolism. FTO is the first RNA dioxygenase shown to have an ‘eraser’ function involved in demethylation of m⁶A in mRNA in the cell (48), raising the possibility that m⁶A is reversible. Recent work showed that FTO has a strong demethylation activity for m⁶Am at the 5΄ termini of capped mRNAs (49). ALKBH5 is another eraser for m⁶A in mRNA (50). ALKBH8, a unique protein containing an AlkB domain fused to a tRNA methyltransferase domain, is involved in biogenesis of 5-methoxycarbonylhydroxymethyluridine (mchm⁵U34) at the wobble position of ct-tRNAs (51,52). ALKBH3 has demethylation activity toward internal m¹A in mRNA (53,54), and recent work showed that ALKBH3 can demethylate m⁶A in tRNAs (55), though exact position of m⁶A in tRNA has not been determined yet.

ALKBH1, the first mammalian homolog of E. coli AlkB to be identified (44), is widely conserved among vertebrates and arthropods. ALKBH1 has demethylation activity toward m³C in single-stranded DNA and RNA in vitro (37). ALKBH1 was once proposed to regulate neurodevelopment by demethylating histone H2A (33); however, this demethylation activity was not confirmed (16). Instead, it was proposed that ALKBH1 has the ability to demethylate m⁶dA in DNA (16). A study published last year showed that ALKBH1 has a role in demethylating m¹A58 in ct-tRNAs (17), thus regulating protein synthesis by downregulating translation initiation and decreasing participation of tRNAs in the elongation stage. The physiological role of ALKBH1 has been inferred based on phenotypic features of Alkbh1-null mice. A mouse harboring a deletion of exon 3 exhibited intra-uterine growth retardation with defective placenta (56). By contrast, the exon 6 deletion mouse exhibited non-Mendelian inheritance of the targeted Alkbh1 allele and sex-ratio distortion of the offspring (57), indicating that ALKBH1 plays critical roles in the early development. Our finding of ALKBH1 as an RNA dioxygenase responsible for f⁵C formation in cytoplasmic and mitochondrial tRNAs might provide insight into the enzyme's physiological role in mammals.

In this study, we showed that human ALKBH1 catalyzes hydroxylation and oxidization of m⁵C34 in both ct-tRNA^Leu(CAA) and mt-tRNA^Met. These findings demonstrated that nuclear ALKBH1 and mitochondrial ALKBH1 play distinct roles in tRNA modifications. Although we elucidated the biogenesis of f⁵Cm34 and hm⁵Cm34 in ct-tRNA^Leu(CAA), the functions of these modifications remain obscure. Based on the functional importance of f⁵C34 in mt-tRNA^Met, f⁵Cm34/hm⁵Cm34 might contribute to decoding of UUA as well as UUG. These modifications are thought to contribute to robust decoding of UUA, together with an isoacceptor tRNA^Leu bearing a modified uridine at the wobble position (U*34), which is responsible for decoding UUR. In our RNA-MS analyses (Figures 2B and 3D), we measured the exact frequency of the wobble modifications in ct-tRNAs^Leu(CAA) isolated from mouse fetus and HEK293T cells (Supplementary Table S2), and observed that modification status differed between these two tRNAs. Mouse fetus ct-tRNAs^Leu(CAA) (mature form) contained 14% f⁵Cm34 and 79% hm⁵Cm, whereas human ct-tRNAs^Leu(CAA) isolated from HEK293T had 30% f⁵Cm34, 26% hm⁵Cm34 and 34% hm⁵C34. These results suggest that the ratio of f⁵Cm34 and hm⁵Cm34 can be altered in various tissues and cells, indicating that their modification status is dynamically regulated in various physiological contexts. If f⁵Cm34 and hm⁵Cm34 play distinct roles in decoding, cytoplasmic protein synthesis could be regulated by the alterative wobble modifications. In addition, we found that f⁵C34 is 2΄-O-methylated more robustly than hm⁵C34 (Supplementary Table S2). Considering that FTSJ1 is predominantly localized in the cytoplasm (34) (The Human Protein Atlas, http://www.proteinatlas.org/), precursor tRNA^Leu(CAA) bearing f⁵C34 might be exported to the cytoplasm more efficiently than the tRNA bearing hm⁵C34. Otherwise, FTSJ1 and its partner protein (probably WDR6), which determines specificity to position 34, might prefer f⁵C34 over hm⁵C34 for 2΄-O-methylation. Pathogenic mutations in FTSJ1 and NSUN2 are associated with intellectual disabilities (58–62). Thus, dysregulation of protein synthesis caused by loss of f⁵Cm34/hm⁵Cm34 in ct-tRNA^Leu(CAA) might contribute to molecular pathogenesis of these diseases.

In the case of ct-tRNA^Leu(CAA), NSUN2 introduces m⁵C34 to initiate formation of f⁵Cm34/hm⁵Cm34. By contrast, NSUN3 forms m⁵C34 to initiate f⁵C34 formation in mt-tRNA^Met. However, we found that hydroxylation and oxidation of m⁵C34 in both tRNAs are commonly catalyzed by ALKBH1. ALKBH1 KO cells exhibited slow growth in galactose medium, reduced Complex I activity and low oxygen consumption, indicating that the physiological role of ALKBH1 is connected with mitochondrial function. Because ALKBH1 KO cells exhibited phenotypes similar to those of NSUN3 KO cells, loss of f⁵C34 is likely to be a primary cause of mitochondrial dysfunction of ALKBH1 KO cells. Both NSUN3 KO and ALKBH1 KO cells exhibited Complex I deficiency, indicating that f⁵C34 in mt-tRNA^Met is required for efficient mitochondrial translation of protein components for Complex I. In fact, we also observed severe reduction of mitochondrial translation in ALKBH1 KO cells. In support of this finding, steady state levels of ND2 and ND5 decreased, and assembly of Complex I was defective, in NSUN3 KO cells (6). Because mt-tRNA^Met contains m⁵C34 in ALKBH1 KO cells, m⁵C34 appears to have no ability to decode AUA, as observed for C34 in NSUN3 KO cells.

Previous studies reported that mt-tRNA^Met contains m⁵C34 in human culture cells. Haag et al. (15) performed bisulfite sequencing of total RNA from HeLa cells with or without NaBH₄ treatment, and found that a majority of the modification is f⁵C34, whereas m⁵C34 is actually present in mt-tRNA^Met in HeLa cells. Van Haute et al. (13) used fCAB RNA-seq and RedBS RNA-seq to estimate that mt-tRNA^Met isolated from human fibroblast contained 36–38% f⁵C34 and ∼30% m⁵C. Because both reports analyzed modification status by indirect methods, which may suffer from bias resulting in erroneous measurement of the frequency of each modification, we assume that the abundance of m⁵C34 in mt-tRNA^Met was overestimated in both cases. Otherwise, m⁵C34 might have been detected in primary transcripts or precursors of mt-tRNA^Met. We isolated mt-tRNA^Met from HEK293T and HeLa cells, and directly analyzed anticodon-containing fragments by LC/MS to determine the exact frequency of each modification. We could detect the RNA fragments containing C34, m⁵C34, hm⁵C34,or f⁵C34 separately, based on their exact molecular masses. RNA fragments are ionized by ESI as negative ions derived from phosphate groups. Hence, ionization efficiencies of RNA fragments bearing the same sequences, but different modifications, do not significantly differ in general, because ESI ionization relies mainly on numbers of phosphate groups, rather than the type of base modifications (23). The relative amounts of these fragments can be calculated by their detection intensities. Using this method, we demonstrated that mt-tRNA^Met is fully modified with f⁵C34 in both HEK293T (Figure 4A) and HeLa cells (Supplementary Figure S3). No hypomodified fragment bearing C34, m⁵C34 or hm⁵C34 was detected in mature mt-tRNA^Met, which participates in translation. However, we cannot rule out the possibility that f⁵C34 is dynamically regulated in different tissues and cells via alternative expression of NSUN3 and ALKBH1, or due to an intracellular change in concentrations of AdoMet, Fe²⁺, 2-OG and O₂.

In vitro reconstitution of f⁵C34 revealed that ALKBH1 catalyzes hydroxylation and oxidation of m⁵C34 in the presence of 2-OG and Fe²⁺ (Figure 6A). In addition, we successfully reconstituted f⁵C34 from C34 by consecutive reactions catalyzed by NSUN3 and ALKBH1. Over the course of these reactions, m⁵C34 was formed rapidly, followed by the appearance of hm⁵C34 and ultimately f⁵C34 gradually accumulated, but no 5-carboxycytidine (ca⁵C34) was formed. This reaction profile indicates that ALKBH1 catalyzes two-step reactions initiated by hydroxylation of m⁵C34 to form hm⁵C34, and followed by oxidation of hm⁵C34 to form f⁵C34. Haag et al. (15) only detected f⁵C34 as an oxidation product of m⁵C34 in ASL in vitro and did not detect any hm⁵C34 as an intermediate. This discrepancy might arise due to differences in our assay conditions or recombinant protein constructs. In genomic DNA, the TET family enzymes, another family of Fe²⁺/2-OG-dependent oxygenases, hydroxylate m⁵dC to form hm⁵dC as a major product (63,64). TET enzymes can also oxidize m⁵C in RNA to form hm⁵C in vitro as well as in human culture cells (65). In Drosophila, hm⁵C in RNA is synthesized by dTet (66), indicating that TET enzymes also participate in the formation of hm⁵C in RNA. However, hm⁵Cm in ct-tRNA^Leu(CAA) was completely absent in the ALKBH1 KO cells used in this study, ruling out a possibility that TET enzymes are redundantly involved in this process. Considering that the target sites of ALKBH1 reside in loop regions of RNA hairpins (17), ALKBH1 might oxidize m⁵C to form hm⁵C or f⁵C in the loop regions of mRNAs.

We also investigated whether ALKBH1 is involved in demethylation of m¹A in mitochondria and found that the m¹A frequency at two sites (m¹A16 in mt-tRNA^Arg and m¹A58 in mt-tRNA^Lys) was elevated (Figure 7), whereas four other sites in mt RNAs were unchanged, upon ALKBH1 KO (Supplementary Figure S2A–C and G). m¹A16 and m¹A58 are found in the loop regions of the D-stem and T-stem, respectively. No m¹A at position 9 was targeted by ALKBH1. Taken together with the formation of f⁵C34, these results indicate that ALKBH1 prefers to recognize the stem-loop structure of RNA and oxidize the target methyl group. We previously reported that m¹A58 in mt-tRNAs is introduced by Trmt61B (27). However, knockdown of this gene in HeLa cells caused no obvious phenotype. Thus, regulation of m¹A58 by ALKBH1 might have a moderate effect in mitochondrial function, and the mitochondrial dysfunction observed in ALKBH1 KO cells should be largely attributed to the loss of f⁵C34 in mt-tRNA^Met.

In this study, we found that ALKBH1 is responsible for biogenesis of hm⁵Cm34/f⁵Cm34 in ct-tRNA^Leu(CAA), as well as f⁵C34 in mt-tRNA^Met. ALKBH1 KO cells exhibited strong reduction in mitochondrial translation and reduced respiratory complex activities, demonstrating that ALKBH1-mediated f⁵C34 formation is required for mitochondrial functions. In vitro reconstitution of f⁵C34 revealed that ALKBH1 first hydroxylates m⁵C34 to form hm⁵C34, and then oxidizes hm⁵C34 to form f⁵C34. We also found two m¹A sites in mt-tRNAs that were present at higher frequencies in ALKBH1 KO cells, indicating that mitochondrial ALKBH1 is also involved in m¹A demethylation.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); Japan Society for the Promotion of Science (JSPS) [26113003 and 26220205 to Ts.S., 26702035 to Ta.S.]. Funding for open access charge: JSPS; Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Conflict of interest statement. None declared.