Fig. 3. Nsun2 controls Th17 cell generation via m5C-mediated mRNA stability.

a Heatmap showing differentially expressed genes in Th17 cells between wild-type and Nsun2^cKO mice. b IGV tracks displaying ATAC-seq, RoRγt ChIP-seq (GSE40918), mRNA-seq, Nsun2 RIP-seq (IP and input) and m⁵C MeRIP-seq (IP and input) enrichment near Il17a. c–f Verification experiment of rescuing wild-type (Nsun2-WT) and methyltransferase activity deficient mutant (Nsun2-C321A) of Nsun2 in Th17 cells. Sorted naive CD4⁺ T cells from wild-type and Nsun2^cKO mice respectively were activated for 1 day, then infected with retrovirus (RV)-mediated Nsun2-WT or Nsun2-C321A expression following induced under Th17-inducing condition for another 3 days. Vector as control. Cell lysates were carried out western blot (c). mRNA level of Il17a was detected by RT-qPCR (n = 3 per group) (d), and Th17 differentiated frequency was analyzed by FACS (e), and summary of the frequency of Th17 cells (n = 3 per group) (f). g The half-lives of Il17a in Th17 cells of wild-type and Nsun2^cKO were detected by RT-qPCR. The residual RNAs were normalized to the values at 0 min (n = 3 per group). h Schematic representation of the synthesized reporter RNA based on Il17a mRNA. Il17a-C and Il17a-m⁵C contained 15 nt of GFP sequence and 63 nt of Il17a RNA with or without a m⁵C modification site at 424 of cytosine on Il17a mRNA, and the definite sequence information was shown in Supplementary Data 7. i The RNA abundance of Il17a-m⁵C (Red) and Il17a-C (Gray) in Th17 cells were detected by RT-qPCR, and normalized to the value at 0 min. Gapdh serves as an internal control (n = 3 per group). All data are representative of at least two independent experiments. Data are analyzed using two-tailed unpaired Student’s t-test. Error bars represent mean ± s.e.m (d, f, g and i). Source data are provided as a Source Data file.