RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

• Published in: Cellular and molecular life sciences : CMLS Vol. 75; no. 8; pp. 1483 - 1497
• Main Authors: Trixl, Lukas, Amort, Thomas, Wille, Alexandra, Zinni, Manuela, Ebner, Susanne, Hechenberger, Clara, Eichin, Felix, Gabriel, Hanna, Schoberleitner, Ines, Huang, Anming, Piatti, Paolo, Nat, Roxana, Troppmair, Jakob, Lusser, Alexandra
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.04.2018; Springer Nature B.V
• Subjects: 5-Methylcytosine - metabolism; Animals; Biochemistry; Biomedical and Life Sciences; Biomedicine; Catalysis; Cell Biology; Cell Differentiation; Cell fate; Cell Line; Cell proliferation; Cell self-renewal; Cytosine; Deactivation; Differentiation (biology); Electron transport; Embryo cells; Embryoid Bodies - cytology; Embryoid Bodies - enzymology; Endoderm; Gene expression; Gene Expression Regulation, Developmental; Genes, Reporter; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Inactivation; Life Sciences; Methylation; Methyltransferases - genetics; Methyltransferases - metabolism; Mice; Mitochondria; Mitochondria - enzymology; Mitochondria - genetics; Mouse Embryonic Stem Cells - cytology; Mouse Embryonic Stem Cells - enzymology; Neural Plate - cytology; Neural Plate - enzymology; Neural Plate - growth & development; Neuroectoderm; Original; Original Article; Oxidation; Oxidative Phosphorylation; Phosphorylation; Pluripotency; Reactive oxygen species; Reactive Oxygen Species - metabolism; Ribonucleic acid; RNA; RNA, Transfer, Met - genetics; RNA, Transfer, Met - metabolism; Signal Transduction; Signaling; Somatic cells; Stem cell transplantation; Stem cells; Transcriptome; Translation; Mitochondria; Self-renewal; tRNA modification; Neuroectoderm; 5-Methylcytosine; Bisulfite sequencing; Epitranscriptome
• ISSN: 1420-682X; 1420-9071
• DOI: 10.1007/s00018-017-2700-0

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 tRNA Met . 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-tRNA Met 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.