RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity
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Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.
KeywordstRNA modification 5-Methylcytosine Mitochondria Bisulfite sequencing Neuroectoderm Self-renewal Epitranscriptome
RNA modifications have been shown to play important roles in the function and metabolism of all types of RNA in eukaryotes and in bacteria. Particularly, tRNAs are extensively modified, which is reflected in the fact that they carry approximately 90 out of the ~ 150 known RNA modifications . The pattern of tRNA modification ranges from highly conserved types of modifications at highly conserved nucleosides to modifications that are unique to specific residues of (a) specific tRNA (group) . Consistent with the great diversity of modifications, they are involved in a wide range of functions. tRNA modifications are required for proper tRNA folding, they can affect aminoacylation, regulatory tRNA cleavage, as well as interaction with and decoding of the mRNA during translation [3, 4, 5, 6]. Stress-induced changes in modification patterns regulate tRNA cleavage, which, in turn, results in downregulation of protein biosynthesis to allow for the repair of cell damage or apoptosis and might, therefore, be important for the modulation of cancer cell metabolism . tRNA modification defects have also been associated with mitochondrial disease [4, 8]. For instance, loss of the taurine modification at position U34 in the anticodon loop of human mt-tRNALeu(UUR) was detected in patients suffering from MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome . 5-Taurinomethyluridine was shown to stabilize wobble pairing with codons that end in a G . Another mitochondrial anticodon loop modification is 5-formylcytidine (f5C) at position C34 of mt-tRNAMet, which stabilizes base-pairing with A to enable decoding of the non-universal mitochondrial AUA in addition to the universal AUG codon [10, 11]. Recently, four studies have identified the enzymes that are responsible for the generation of f5C in mt-tRNAMet. It was shown that the RNA cytosine methyltransferase (RCMT) NSUN3 targets C34 for carbon 5 methylation in human cells [12, 13, 14], while the alpha-ketoglutarate and Fe(II)-dependent dioxygenase ALKBH1/ABH1 further oxidizes 5-methylcytidine (m5C) to f5C [14, 15]. NSUN3 deletion abolished f5C in tRNAMet of human dermal fibroblasts, HeLa, and HEK293 cells and led to impaired mitochondrial translation efficiency presumably by interfering with efficient decoding of the AUA codons in mitochondrially encoded transcripts of electron transport chain components [12, 13, 14]. Functional inactivation of NSUN3 as well as point mutations found in patients that occur in the vicinity of C34 on mt-tRNAMet and affect NSUN3-mediated methylation resulted in mitochondrial disease [12, 13].
Adult somatic cells rely heavily on oxidative phosphorylation (OXPHOS) in mitochondria to meet their energy demands. Therefore, defects in the electron transport chain typically have severe consequences for cell metabolism. By contrast, embryonic stem cells (ESCs) predominantly utilize anaerobic glycolysis, and it has been demonstrated that their mitochondria show reduced respiration, they have globular shape and perinuclear localization [16, 17]. Reprogramming of somatic cells to pluripotent stem cells is accompanied by morphological changes of mitochondria and a downregulation of electron transport chain complex I and II subunits . Nevertheless, although mitochondria in stem cells may not be essential for ATP production, they appear to support stemness by enforcement of alternative pathways, such as threonine catabolism in murine but not human ESCs or by channelling intermediates from the tricarboxylic acid cycle for anabolic pathways . Differentiation of ESCs, on the other hand, is accompanied by a shift from glycolytic to oxidative metabolism reflected in a gain in mitochondrial mass, upregulation of mitochondrial enzymes and downregulation of glycolytic enzymes, increased oxygen consumption, and lower lactate production. ESC differentiation is also affected by mitochondrial reactive oxygen species (ROS), although the exact mechanisms in ESCs are not well understood .
Given the impact of C34 modification in mt-tRNAMet on mitochondrial translation of electron transport chain components in human somatic cells [12, 13], we examined if C34 modification also plays a critical role in mouse ESCs despite their favouring anaerobic glycolysis over OXPHOS. We catalytically inactivated the C34 methyltransferase Nsun3 in mouse ESCs by CRISPR/Cas9 and examined the functional consequences on ESC self-renewal, stemness, energy metabolism, and differentiation potential.
Materials and methods
Embryonic stem cell culture and differentiation
Mouse embryonic stem cells (129/Sv) were cultured in ESC medium (LIF+2i) (DMEM high glucose with GlutaMAX-1 [Gibco], 20% FBS [Gibco], 1 × non-essential amino acid mix [Gibco], 0.05 mM β-mercaptoethanol, 10 µg/ml LIF [Sigma], 3 µM CHIR99021, 1 µM PD0325901 [both Axon Medchem]) in gelatine-coated culture dishes at 37 °C and 5% CO2. Induction of embryoid body (EB) formation and EB outgrowth were performed as previously described . Differentiation of ESCs into the ectodermal lineage was performed as described previously . In brief, ESCs were cultured in N2B27 supplemented serum-free medium, containing 10 µg/mL LIF, 3 µM CHIR99021, and 1 µM PD0325901 for 24 h in 25 cm2 flasks before passaging to 6-well plates in the same medium containing only 0.4 µM PD0325901 for 2 days. After that, cells were incubated with 1 µM of LDN193189 (BMP antagonist; Sigma) for additional 4 days.
Catalytic inactivation of Nsun3 in mouse ESCs
To generate an ESC cell line expressing catalytically inactive Nsun3, the CRISPR/Cas9 method was used . A double-stranded oligo containing the sgRNA sequence targeting the catalytically important T264C265 motif encoded in exon 6 of mouse Nsun3 (NC_000082.6) was cloned into the vector pX458 , which encodes GFP in addition to the Cas9 nuclease. The recombinant plasmid was transfected into ESCs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and cultured for 24 h. Cells were then trypsinized and subjected to FACS sorting of single GFP+ cells into 96-well plates containing 200 µl of a 1:1 mixture of preconditioned and fresh ESC medium. After about 6 days, ES cell colonies were visible. Several colonies were expanded and screened for indel mutations in Nsun3 exon 6. To this end, PCR (NSUN3fw 5′ AGCTTTGCCCTTTTCCGGAA and NSUN3rev 5′ CGTGCTGTGATGATCCCCAA) was performed to amplify the region around the targeting site using the Terra PCR Direct Polymerase Mix (Clontech). Genomic DNA extraction and PCR were performed according to the manufacturer’s instructions. PCR products were subcloned into pGEM-T Easy vector (Promega) and sequenced.
Nsun3-GFP construct generation, transfection, and Nsun3 localization
The pX458 vector  was digested with AgeI and BsrGI restriction enzymes. The Nsun3 ORF was amplified from mouse cDNA using the following primers: NSUN3_fw: 5′ TCA CTT TTT TTC AGG TTG GAG CCA CCA TGC TGA CTC GGC TGA AAG 3′ and NSUN3_rev: 5′ CCT TGC TCA CTG GCT TAC AAA ATT CGC C 3′. The GFP ORF was amplified from a pMT_EGFP plasmid  with the following primers: GFP_fw: 5′ TTG TAA GCC AGT GAG CAA GGG CGA GGA G 3′ and GFP_rev: 5′ AGC TCT AGT TAG AAT TCC TTT CAC TTG TAC AGC TCG TCC 3′. Both fragments and the vector were fused using the NeBuilder (NEB) system to generate a construct for the expression of Nsun3 with a C-terminal EGFP tag. The plasmid was transfected into mouse ESCs using Lipofectamine 2000 (Thermo Fisher Scientific) following the standard methods. 24 h post-transfection, mESCs were seeded into 8-well chambered Nunc Lab-Tek coverglasses and stained using 100 nM MitoTracker® Red CM-H2XRos (Invitrogen) in serum-free DMEM for 30 min at 37 °C in a 5% CO2 environment. Afterwards, medium was replaced by complete ESC medium and live cell images were taken using a Leica SP5 confocal microscope. For Alkbh1 localization, mESCs were transfected with 4 µg of the pX-Nsun3-GFP plasmid using lipofectamine. 24 h post-transfection, cells were detached by Accutase® (Sigma) to obtain single cells and subsequently allowed to settle down on cover slips. Fixing, antibody incubation and washing was performed exactly as described before . Antibodies used: anti-GFP (1:10,000; Thermo Fisher, A-11122) and anti-ALKBH1 (1:100; Abnova, H00008846-B01P). DAPI was used to visualize DNA. Cell images were taken as described above. Images were processed using ImageJ and Affinity Photo (Serif) software.
ESC proliferation measurements
Cell proliferation was determined using the CyQuant® Cell Proliferation Assay Kit (ThermoFisher) according to the manufacturer’s instructions. 3000 cells were seeded in five technical replicates in 96-well plates in ESC medium. Every 24 h, cells were harvested, washed in PBS, and frozen at − 70 °C. At the end, all cell samples were resuspended in 200 µl each of CyQuant® GR dye/cell-lysis buffer and fluorescence was measured using an FLUOstar® Omega microplate reader at 480 nm setting. Fluorescence values were converted to cell numbers using the linear equation of a standard curve that was generated by measuring serial dilutions of a known number of cells.
RNA extraction, RT-qPCR, and strand-specific Northern blotting
RNA extraction and reverse transcription real-time PCR (RT-qPCR) were performed essentially as described previously  on cDNA obtained from at least three independent biological replicates. Primer sequences are available upon request. Statistical significance of differences between mutant and wild-type samples was determined using multiple unpaired t test with Holm–Sidak correction for multiple testing (Graphpad Prism 7.0). Expression levels of mitochondrially encoded transcripts were analyzed by separating 5 µg total RNA on 1.2% agarose/1.1% formaldehyde gels, blotting onto Hybond-N membrane (GE Healthcare) and hybridizing with DIG-labelled strand-specific RNA probes in “High-SDS” solution (7% SDS, 50 mM Na-phosphate buffer, pH 7.0, 50% deionized formamide, 5 × SSC, 2% Roche blocking solution, 0.1% Na-lauroylsarcosine). Since both strands of the mitochondrial genome are transcribed, strand-specific ssRNA probes were generated by in vitro transcription of PCR amplified fragments of mitochondrial transcripts using the MEGAScript T7 in vitro transcription kit (Thermo Fisher Scientific) in combination with the DIG RNA labelling mix (Roche) according to the manufacturer’s instructions. After hybridization, northern blots were subjected to stringent washes in 0.1 × SSC/0.1% SDS at 68 °C and finally to DIG detection following the Roche protocol. Chemiluminescent signal was captured using a Fusion SL3500 WL instrument (Vilber) or X-ray film. Signal intensities were quantified using the Image Studio Lite (v5.2) software. Statistical significance of differences was calculated by multiple unpaired t test analysis in Graphpad Prism 7 (*p < 0.05).
Reductive treatment of RNA and RNA bisulfite sequencing
To reduce 5-formylcytosine to 5-hydroxmethlycytosine, RNA was treated with sodium borohydride following a previously described method . Briefly, 1.5 µg of total RNA in 15 µl were mixed with 5 µl of a freshly prepared 1 M NaBH4 solution (in water) and incubated for 30 min at room temperature in the dark. The reaction was stopped by addition of 10 µl 750 mM sodium acetate (pH 5) and kept at room temperature until no further gas was released. Samples were then subjected to PCR-mediated bisulfite sequencing exactly as described before . cDNA synthesis was performed with an mt-tRNAMet-specific stem loop primer (5′ CTCAACTGGTGTCGTGGAGTCGGCAAT TCAGTTGAGTGGTTAAACCAAC); mt-tRNAMet was then amplified with primers DtRNAm fw 5′ AAGGTTAGTTAATTAAGTTATT and UniSL rev 5′ CACGACACCAGTTGA, subcloned into pGEM-T vector (Promega), and inserts were subjected to Sanger sequencing.
Analysis of mitochondrial translation by metabolic labelling
Pulse labelling of mitochondrial proteins was performed as described before , with the following changes: 9.5 × 105 wild-type and Nsun3-mutant ES cells per well were seeded into a gelatine-coated 6-well plate and cultivated for 24 h. Cells were washed once with PBS followed by one wash with labelling medium (ESC medium without cysteine and methionine) and a 30 min incubation in 1.8 ml labelling medium. Then, 200 µl of emetine (1 mg/ml) were added and incubated at 37 °C for 40 min to stop cytoplasmic translation. To label mitochondrially translated products, 40 µl of l-[35S]-methionine (10 mCi/ml; Hartman Analytic) were added and the cells were incubated for 1 h. Cells were washed in standard ESC medium and incubated another 10 min at 37 °C. Cells were then harvested by trypsinizing, washed once with ice-cold PBS, and extracted with ice-cold RIPA buffer containing protease-inhibitors. Proteins were fractionated by gel electrophoresis in 16% Tricine gels (Invitrogen) and stained with Coomassie brilliant blue, and radioactive signals were visualized by phosphoimaging. Signal intensities were quantified using the Image Studio Lite (v5.2) software. Statistical significance of differences was calculated by multiple unpaired t test analysis in Graphpad Prism 7 (*p < 0.05).
Real-time PCR-based determination of mitochondrial DNA content
DNA was extracted from wild-type and Nsun3 cat/cat cells using standard protocols. 100 ng of DNA was subjected to real-time PCR to amplify a specific region in nuclear DNA (apoBfw 5′ CGTGGGCTCCAGCATTCTA and apoBrev 5′ TCACCAGTCATTTCTGCCTTTG) and two regions in the mitochondrial genome (Mito1_ND5: ND5fw 5′AATAGTGACGCTAGGAATAA and ND5rev 5′GATGTCTTGTTCGTCTGCCA; Mito2_ Rnr2: Rnr2fw 5′AGGGATAACAGCGCAATCCT and Rnr2rev 5′AGGGATAACAGCGCAATCCT). Mitochondrial amplification signals were normalized against the nuclear signal and expressed relative to wild-type ESCs. Mean values ± SEM of three experiments are shown and statistical significance was calculated by unpaired t test (*p < 0.05).
Respiration and glycolysis measurements
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined with a Seahorse XFe96 Analyzer (Seahorse Bioscience). To this end, 40,000 wild-type or mutant ESCs were seeded into 7–8 wells of a gelatine-coated 96-well analyzer plate (Seahorse Bioscience) and incubated overnight under standard conditions. For determination of OCR, oligomycin (2 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (0.6 µM), and rotenone/antimycin A (0.5 μM) were injected according to the XF Cell Mito Stress Test Kit (Seahorse Bioscience). Glycolytic function was determined using the XF Glycolysis Stress Test Kit (Seahorse Bioscience). Glucose (10 mM), oligomycin (2 µM), and 2-deoxy-glucose (2-DG) (50 mM) were injected according to the manufacturer’s protocol. The obtained OCR (respiration) and ECAR (glycolysis) data were normalized against cell numbers in each well and analyzed using the Wave software as well as the XF Report Generator (Seahorse Bioscience). Cell numbers were determined by Hoechst 33342 (Thermo Scientific) staining combined with fluorescence measurement using Infinite F200 PRO instrument (Tecan).
ROS and mitochondrial membrane potential measurements
Mitochondrial ROS were imaged by fluorescence microscopy after staining the cells with 100 nm MitoTracker Red CM-H2XROS (Thermo Fisher Scientific) in serum-free DMEM medium. For this purpose, 50,000–80,000 cells/well were seeded in the 8-well Nunc Lab-Tek II Chamber Slide System (Thermo Fisher Scientific). For ROS measurements in cells undergoing differentiation, varying starting cell numbers were seeded into the individual wells depending on the duration of culture: 50,000–80,000 (15 h), 25,000–35,000 (28 h), and 10,000–15,000 cells/well (48 h) in ESC medium with LIF and 2i. After 16 h cells were either left untreated or differentiation was induced by medium without LIF and 2i. ROS staining was performed for 30 min at 37 °C without stress or after stressing the cells by incubation in 1 mM H2O2 in ESC medium for 30 min at 37 °C. Digital images were taken using an Olympus IX-70 inverted microscope (Olympus America) with an Olympus 40 × water immersion objective (numerical aperture 0.8) and an Olympus U-RFL-T mercury-vapor lamp. Image acquisition was performed with a Kappa ACC1 camera and Kappa ImageBase software (Kappa Opto-electronics). For MitoTracker Red CM-H2XROS, a 568-nm filter was used. Gray values were measured using the Scion Image software for Windows. For every experimental condition, gray values from 100 to 120 cells were averaged.
Total cellular ROS levels were determined by staining with 2′,7′-dichlorofluorescin-diacetate (DCF-DA) (Sigma-Aldrich) after stress application as described above. Cells were loaded with 10 μM DCF-DA and incubated for 15 min at 37 °C before FACS measurement. Quantitative analysis was done using the CellQuest software for FACSCalibur (BD Biosciences).
To analyze mitochondrial membrane potential, control or H2O2-stressed cells were incubated for 15 min at 37 °C with 25 nM tetramethylrhodamine methyl ester (TMRM, Invitrogen). TMRM fluorescence was detected by FACS. In control experiments, dissipation of membrane potential was observed after addition of 5 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma-Aldrich). Values were expressed as geometric means after correction for background TMRM fluorescence measured in uncoupler (FCCP)-treated cells.
Statistical analyses were performed in Graphpad Prism 7 using one-way ANOVA with Bonferroni correction.
Generation of ESCs with catalytically inactive Nsun3
Formylation of C34 is carried out by ALKBH1 [14, 15], and like NSUN3, ALKBH1 localized to mitochondria in HEK293 cells . Consistent with this, co-staining of Alkbh1 with GFP-tagged Nsun3 in mouse ESCs revealed a clear although not perfect overlap suggesting mitochondrial localization of Alkbh1 (Fig. 1a). This result contrasts the finding by Ougland et al. for human ESCs, where ALKBH1 was shown to be predominantly present in the nuclei but not in mitochondria [31, 32]. It is possible that mouse and human ESCs are different with respect to ALKBH1 localization. However, we consider it likely that a small portion of Alkbh1 is also present in nuclei and/or in the cytoplasm of mouse ESCs or of other cell types, since it was recently found to be involved in demethylation of cytoplasmic m1A-modified tRNAs and in the oxidation of m5C34 of cytoplasmic tRNALeu in HEK293 and HeLa cells [15, 33].
Besides m5C34, we detected a previously unknown methylation at C47 in the variable loop of mt-tRNAMet that is not conserved in human mt-tRNAMet. The extent of C47 methylation was virtually complete not only in ESCs but also in differentiating ESCs, adult brain tissue and in MEFs (Fig. 1c, Supplementary Fig. S1). Consistent with Nsun3 specifically targeting C34, C47 methylation did not change in the Nsun3-mutant ESCs (Fig. 1c). Thus, while wild-type ESCs possess methylated and formylated C34 in mt-tRNAMet, Nsun3-mutant cells exhibit loss of C34 methylation and strong reduction of formylation indicating that we have generated a cell line expressing a catalytically inactive Nsun3 protein (hereafter termed Nsun3cat).
Nsun3cat affects proliferation but not pluripotency marker expression in ESCs
Catalytically inactive Nsun3 compromises mitochondrial activity
The elevated steady-state mitochondrial transcript levels in Nsun3 cat/cat cells might originate from an increase in mitochondrial biogenesis in the mutant cells to compensate for the compromised protein translation. Indeed, when we measured mitochondrial DNA relative to nuclear DNA by real time PCR, we found a ~ 1.5-fold increase of mitochondrial DNA in Nsun3 cat/cat ESCs compared to wild-type ESCs (Fig. 3f). The observed upregulation of mitochondrial transcripts and DNA content correspond well with the effects observed upon deletion of other factors that compromise mitochondrial translation (e.g. [34, 35]).
Nsun3 inactivation skews differentiation of ESCs towards mesendoderm
The strongest dysregulation, however, was observed for markers of the neuroectoderm. Sox1 and Pax6 showed dramatically lower levels in Nsun3 cat/cat EBs at all stages but most pronounced at days 2–6 of outgrowth (Fig. 6c). Consistently, the pluripotency factor Sox2, which is also expressed in neuroectodermal cells , was significantly downregulated in Nsun3-mutant EBs at day 2 compared to wild type (adjusted p = 0.04; Supplementary Fig. S2). These results may indicate that inactivation of Nsun3 favours differentiation along the meso- and endodermal lineages at the expense of neuroectoderm differentiation programs. To further investigate this idea, we induced neurodifferentiation in Nsun3 cat/cat and wild-type ESCs following published protocols . Similar to the EB differentiation experiments, the expression of Sox1 and Pax6 was downregulated in Nsun3 cat/cat cells compared to wild-type cells, supporting a delay in neuroectoderm differentiation of Nsun3-mutant cells (Fig. 6d). We conclude that ESC differentiation is affected by decreased mitochondrial activity caused by Nsun3-inactivation.
Nsun3 mutation affects Wnt signalling and production of mitochondrial ROS in ESCs
Because Wnt3 was overexpressed in Nsun3 cat/cat cells at the earliest stages of differentiation, we examined Wnt3 expression in ESCs. We found significantly elevated Wnt3 mRNA levels already in Nsun3 cat/cat compared to wild-type ES cells (Fig. 7b), suggesting that Nsun3-mutant ESCs are already primed towards the mes/endodermal lineage without losing pluripotency. To explain the connection between Wnt3 upregulation and compromised mitochondrial translation caused by Nsun3 inactivation, we considered the possibility that the Wnt signalling pathway may be activated by aberrant levels of reactive oxygen species (ROS) in Nsun3 cat/cat ESCs. The mitochondrial respiratory chain is a significant source of ROS and Wnt signalling is known to be sensitive to ROS . Since reduced mitochondrial translation will affect the integrity of respiratory chain complexes and thus may lead either to increased or to decreased ROS production, we quantified total intracellular ROS levels in wild-type and Nsun3-mutant ESCs by FACS after 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and mitochondrial ROS by fluorescence microscopy after MitoTracker Red CM-H2XROS staining. Surprisingly, we did not find any differences in basal ROS levels in wild-type versus Nsun3 cat/cat ESCs (Fig. 7c, d). However, stressing the cells by treatment with H2O2 showed that mitochondrial ROS production in Nsun3-mutant ESCs was significantly weaker than in the wild type (Fig. 7d), whereas total cellular ROS induction was comparable in both cell lines (Fig. 7c).
Thus, while we cannot directly link the increased Wnt3 levels in mutant ESCs to increased ROS levels, it is possible that the observed differentiation defects may, at least in part, involve the inability of the Nsun3-mutant cells to induce sufficient mitochondrial ROS to orchestrate the appropriate signalling cascades. To test this hypothesis, we measured mitochondrial ROS upon induction of differentiation by removal of LIF and 2i. However, we were unable to detect increased ROS levels either in wild-type or in Nsun3-mutant cells during the course of the experiment (Supplementary Fig. 3a), indicating that if ROS plays a role in differentiating ESCs, the required amounts are likely small or transient and beyond the detection limit of our method. Previously, it was reported that mESCs that exhibit low mitochondrial membrane potential and oxygen consumption preferentially differentiate into the mesodermal lineage . To determine, if the observed precocious upregulation of mesodermal markers in Nsun3-mutant cells correlated with lower mitochondrial membrane potential in the mutant ESCs, we measured membrane potential in Nsun3-mutant and wild-type cells using TMRM (tetramethylrhodamine methyl ester) staining and FACS detection . Overall, we found no differences in median TMRM fluorescence between the two cell lines (Supplementary Fig. 3b). H2O2 challenge reduced TMRM fluorescence significantly and more strongly in Nsun3-mutant than in wild-type cells indicating mitochondrial membrane damage, which is consistent with the data on impaired mitochondrial function shown in Fig. 3. When we looked at the fluorescence (and, therefore, membrane potential) distribution among both cell populations, we found a slightly higher number of cells in the lowest 5% of fluorescence intensity for Nsun3-mutant cells compared to wild-type cells (Supplementary Fig. S3b). This difference, however, was not significant, and, therefore, does not establish a clear correlation between membrane potential defects in Nsun3-mutant cells and their aberrant differentiation behavior.
The recent characterization of NSUN3 as a cytosine methyltransferase for mitochondrial tRNAMet highlighted the importance of RNA modifications for mitochondrial function in human cells [12, 13, 14]. Mutations in the human NSUN3 gene resulting in the expression of a severely truncated protein were associated with mitochondrial dysfunction and mitochondrial disease symptoms in a patient . In this work, we show that catalytic inactivation of Nsun3 in mouse ESCs leads to reduced translation of mitochondrially encoded genes, upregulation of glycolysis and reduced oxygen consumption similar to the phenotypes observed in somatic human cells. Nsun3 activity, however, is also important for embryonic stem cell function and differentiation in the mouse despite the fact that ESCs unlike somatic cells rely mostly on glycolysis rather than oxidative phosphorylation for ATP generation. Nsun3 is the closest relative of Nsun4, which also localizes to mitochondria . Nsun4 methylates C911 in mitochondrial 12S rRNA, yet in addition is required for the assembly of the large and small subunits of the mitoribosome in conjunction with the partner protein Mterf4 . By analogy, it is a possibility that Nsun3, too, may have additional functions apart from its methyltransferase activity.
We also identified a cytosine methylation at position 47 of mitochondrial tRNAMet, which was not affected by Nsun3-inactivation. The methyltransferase Nsun2 is known to catalyze this modification at positions C48/49/50 in cytoplasmic tRNAs . However, to date, Nsun2 is not known to localize to mitochondria , and it has not been detected in mitoproteome studies (http://www.mrc-mbu.cam.ac.uk/impi). Thus, the identity of the mitochondrial tRNAMet C47 methyltransferase remains unknown at present. In cytoplasmic tRNAs, C48/49/50 methylation is thought to confer stability to the three-dimensional structure of the tRNA by participating in non-conventional base-pairing with nucleosides of the D-loop . Moreover, cytosine methylation in the variable loop protects tRNAs from nucleolytic cleavage . Whether this also occurs in mouse mt-tRNAMet or why this nucleotide is not conserved in human mt-tRNAMet remain open questions.
Although we found that Nsun3 expression in self-renewing ESCs is low compared to differentiating stem cells, catalytical inactivation, which results in the absence of C34 methylation and formylation of mt-tRNAMet, significantly reduced mitochondrial translation and the proliferation rate of ESCs. This is consistent with previous results demonstrating that drug-mediated inhibition of mitochondrial function negatively affected ESC proliferation [49, 50], while increased respiration in mESCs can boost cell proliferation . Similarly, compromising mitochondrial translation was found to inhibit proliferation of cultured and, therefore, mostly glycolytic MEF and HeLa cells [52, 53, 54]. The nature of this connection, however, remains unknown . While it was shown in previous studies that treatment with drugs interfering with mitochondrial electron transfer and OXPHOS caused an upregulation of the key pluripotency factors Oct4, Nanog, and Sox2 [49, 56], suggesting reinforcement of stem cell self-renewal, Nsun3 inactivation did not affect the expression levels of these genes. This could be due to the fact that respiration is reduced but not abolished in Nsun3-mutant cells, and no increase in ROS levels is detected, while drug treatment by uncoupling electron transfer from ATP generation or by inhibiting complex III activity resulted in significant amounts of ROS [49, 50].
Our study suggests that Nsun3 is an important factor for ESC differentiation, in particular of neuroectoderm differentiation. Compromised neuronal differentiation was previously observed when mitochondrial activity was inhibited either by drug treatment or by mutations in electron transfer chain components [50, 57, 58]. However, in these cases, the defects in mitochondrial function were much more severe than in Nsun3-mutant cells, in which mitochondrial protein translation and oxygen consumption are only about 50% reduced. Moreover, while increased ROS levels accompanied reduced mitochondrial function in earlier studies , this was not the case in Nsun3-mutant ESCs. On the contrary, even upon oxidative stress induction, Nsun3-mutant cells were not able to produce the same amount of mitochondrial ROS as wild-type cells and their membranes were depolarized more severely. How the mitochondrial defects are connected to aberrant differentiation or to the altered Wnt3 expression level in mutant ESCs remains to be addressed in future studies. The Nsun3cat phenotype, however, attenuated respiratory function without abnormal ROS generation (which is often the case with drug-mediated inhibition of the electron transfer chain), makes this ES cell line a useful tool to study the complex interplay between mitochondrial activity and stem cell pluripotency and differentiation.
The impact of cytosine methylation of tRNAs on stem cell differentiation is not limited to Nsun3. Nsun2 also plays an important role in this process, as its mutation was shown to delay differentiation of skin and testis in mice [59, 60]. Methylation of tRNAs by Nsun2 protects from nucleolytic cleavage [48, 61] and serves to regulate protein translation rates, which appears to be involved in controlling stem cell identity . Thus, cytosine methylation of tRNAs regulating decoding potential or stability of tRNAs is emerging as a critical mechanism in the governing of stem cell fate and function.
Open access funding provided by Austrian Science Fund (FWF). We thank Alice Limonciel for the help with the Seahorse Assay. We acknowledge funding of this project by the Austrian Science Fund P27024-BBL to A.L.
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Conflict of interest
All authors declare that they have no conflict of interest.
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