Fig. 1.

Nsun3 localization and catalytic inactivation of Nsun3 in embryonic stem cells. a Nsun3 and Alkbh1 colocalize in mitochondria of murine ESCs. Cells were transiently transfected with an Nsun3-GFP construct and co-labelled with mitotracker (upper panels) or stained with antibodies against GFP and Alkbh1 (lower panels). DNA was visualized by DAPI staining. b Schematic representation of Nsun3 protein domains. Red boxes, catalytically important areas around PC and TC motifs. Sequencing results of mutated clone shows amino acid changes in both alleles around the catalytically important T₂₆₃C₂₆₄ motif (boxed) and a premature stop codon. c RNA from Nsun3-mutant and wild-type cells was subjected to bisulfite sequencing and NaBH₄-bisulfite sequencing to determine the modification levels of C34 and C47 as illustrated in the schematic representation. Nsun3 ^cat/cat cells lose methylation and formylation at C34 but not methylation at C47 of mt-tRNA^Met. RNA was analyzed by bisulfite sequencing with (+) or without (−) preceding NaBH₄ treatment to reduce 5-formylcytosine to 5-hydroxymethylcytosine