Fig. 1: NSUN6 independently catalyzes high-level m⁵C modification on mRNA substrates in vitro.

a The Venn diagram shows the overlap of NSUN6-mediated mRNA m⁵C sites from different datasets^23,32,35,36. The selected mRNAs with high-level m⁵C modifications, used in the following experiments, were labeled in red. b Flowchart of the in vitro methyl transfer activity assay and RNA mass spectrometry assay. The selected mRNAs, as shown in a, were produced through in vitro transcription using T7 RNA polymerase and subsequently incubated with purified NSUN6 in the reaction buffer. When ³H-SAM was used as the methyl donor, the reaction mixture was spotted onto filter paper, and the amount of radioactive [³H]-methyl-RNA was measured using a liquid scintillation counter. When SAM was used as the methyl donor, the reacted RNA was precipitated, dissolved, and digested into single nucleosides, and the target peak of nucleoside was detected using the RNA mass spectrometry system. c Schematic representation of transcribed tRNA and mRNAs. The candidate mRNAs were approximately 100nt in length, with m⁵C sites located in the middle. The position of m⁵C sites on the transcriptome were indicated by arrows, with the 5’ untranslated region (UTR) labeled in pink, the coding sequence (CDS) in black, and the 3’ UTR in blue. The m⁵C sites were highlighted in red. d The amount of methyl group incorporation into tRNA^Thr(UGU), FURIN, MAPK3, PVRL2, REEP6, TIMM50, and MARCKSL1 after incubation with NSUN6 for eight minutes. e The capacity of tRNA^Thr(UGU) and FURIN to be methylated by NSUN6 and the kinetic parameters of NSUN6 for tRNA^Thr(UGU) and FURIN in the methyl transfer assay. f Mass spectrometry of the nucleosides m⁵C (Q1/Q3 = 258.1/126.1) and A (Q1/Q3 = 268.1/136.2) in six mRNAs—FURIN, MAPK3, REEP6, PVRL2, TIMM50, and MARCKSL1—after incubation with or without NSUN6. Target m⁵C peaks were indicated by black triangles; n.d., not detected. The left vertical axis represents the intensity of A, and the right vertical axis represents the intensity of m⁵C. Data in d, and e are presented as the mean ± SD for three independent experiments. Source data are provided as a Source Data File.