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

Abstract

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.Electronic supplementary materialThe online version of this article (10.1007/s00018-017-2700-0) contains supplementary material, which is available to authorized users.

Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer

“…NSUN3 is localized to mitochondria and is required for the methylation of mitochondrially encoded transfer RNA methionine (mt-tRNA Met ) at the "wobble base C34" within the anticodon loop, and the methylated cytosine is further oxidized to 5fC for normal mitochondrial translation of the respiratory chain complex and oxidative phosphorylation. The mutation of NSUN3 is tightly linked to mitochondrial disease and skewed embryonic stem cell differentiation towards the meso-and endoderm lineages instead of neuroectoderm [57,58]. Another tRNA methylation regulator is NSUN6, which partially resides in the Golgi apparatus and pericentriolar matrix in the cytoplasm and mediates specific methylation on tRNA Cys and tRNA Thr at position C72, which affects tRNAs in a late step in their biogenesis.…”

Post-transcriptional regulation by cytosine-5 methylation of RNA

“…Functional studies in mice and humans showed that loss of NSUN3 and lack of formylated methionine tRNA resulted in reduced mitochondrial translation (Fig. 3D) [21,34,36,67]. In addition, it was shown that catalytic inactivation of Nsun3 in mouse ESCs further caused impaired differentiation into the neuroectodermal lineages [67].…”

“…3D) [21,34,36,67]. In addition, it was shown that catalytic inactivation of Nsun3 in mouse ESCs further caused impaired differentiation into the neuroectodermal lineages [67]. Nonetheless, the mechanistic link between the loss of mitochondrial methionine tRNA formylation and the impairment of differentiation remains unknown.…”

Above the Epitranscriptome: RNA Modifications and Stem Cell Identity

“…Further, the modification at the wobble position of selected mt-tRNAs of the uridine base in taurinomethyluridine is necessary to avoid ribosome stalling at certain AAG (lysine) and UUG (leucine) codons [ 123 ]. The reduction of mt-tRNA m 5 C methylation and formylation impairs the mitochondrial translation and respiration processes [ 124 ].…”

“…Moreover, NSUN3 inactivation attenuated induction of mitochondrial reactive oxygen species (ROS) upon stress, which may affect gene expression programs upon differentiation. These findings indicated NSUN3 as a central regulator of stem cell fate and also provide a model system to study the correlation of mitochondrial function with stem cell pluripotency and differentiation [ 124 ].…”