The RNA m5C methylase NSUN4 protects retinal ganglion cells by regulating m5C methylation in the Hedgehog signaling pathway

Glaucoma, one of the leading causes of blindness worldwide, is characterized by retinal ganglion cell (RGC) damage. NOP2/Sun RNA methyltransferase 4 (NSUN4), a known 5-methylcytosine (m5C) writer, is an essential dual-function mitochondrial protein that facilitates both the process of methylation and the coordination of mitoribosome assembly. However, few studies have focused on its role in RGCs. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq) was subsequently conducted to identify differences in the m5C methylome and gene expression profile in the N-methyl-d-aspartate (NMDA)-induced RGC injury model in vivo . We demonstrated that the significantly hypomethylated mRNAs were significantly enriched in the Sonic Hedgehog (SHH) signaling pathway in the NMDA group and that the mRNA expression of the m5C writer Nsun4 was downregulated. Subsequently, we examined the role of NSUN4 in R28 cells in response to glutamate stimulation. m5C dot blot and MeRIP (real-time fluorescence quantitative PCR) qPCR were employed to screen and validate the molecular mechanism of NSUN4 in glutamate-induced R28 cells through m5C regulation. Functionally, NSUN4 suppressed Ca ²⁺ overload, mitochondrial dysfunction, and apoptosis of R28 cells in vitro . Mechanistically, NSUN4 increased the global mRNA m5C methylation level of Shh , GLI Family Zinc Finger Protein 1 ( Gli1 ) and Gli2 . Our study revealed that NSUN4 can alleviate the death of RGCs, which is associated with increased expression of Shh , Gli1 and Gli2 in the SHH signaling pathway in a m5C-dependent manner in R28 cells. Our findings provide new insights into potential biomarkers of retinal excitotoxity.