NSUN2‐mediated m5C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression

Abstract Background The precise temporal and spatial regulation of N 5 ‐methylcytosine (m 5 C) RNA modification plays essential roles in RNA metabolism, and is necessary for the maintenance of epigenome homeostasis. Howbeit, the mechanism underlying the m 5 C modification in carcinogenesis remains to be fully addressed. Methods Global and mRNA m 5 C levels were determined by mRNA isolation and anti‐m 5 C dot blot in both retinoblastoma (RB) cells and clinical samples. Orthotopic intraocular xenografts were established to examine the oncogenic behaviours of RB. Genome‐wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m 5 C‐methylated RNA immunoprecipitation sequencing (m 5 C‐meRIP‐seq). Organoid‐based single‐cell analysis and gene‐correlation analysis were performed to verify the NSUN2/ALYREF/m 5 C‐PFAS oncogenic cascade. Results Herein, we report that NSUN2‐mediated m 5 C RNA methylation fuels purine biosynthesis during the oncogenic progression of RB. First, we discovered that global and mRNA m 5 C levels were significantly enriched in RBs compared to normal retinas. In addition, tumour‐specific NSUN2 expression was noted in RB samples and cell lines. Therapeutically, targeted correction of NSUN2 exhibited efficient therapeutic efficacy in RB both in vitro and in vivo. Through multiomics analyses, we subsequently identified phosphoribosylformylglycinamidine synthase (PFAS), a vital enzyme in purine biosynthesis, as a downstream candidate target of NSUN2. The reintroduction of PFAS largely reversed the inhibitory phenotypes in NSUN2‐deficient RB cells, indicating that PFAS was a functional downstream target of NSUN2. Mechanistically, we found that the m 5 C reader protein ALYREF was responsible for the recognition of the m 5 C modification of PFAS, increasing its expression by enhancing its RNA stability. Conclusions Conclusively, we initially demonstrated that NSUN2 is necessary for oncogenic gene activation in RB, expanding the current understanding of dynamic m 5 C function during tumour progression. As the NSUN2/ALYREF/m 5 C‐PFAS oncogenic cascade is an important RB trigger, our study suggests that a targeted m 5 C reprogramming therapeutic strategy may be a novel and efficient anti‐tumour therapy approach.