RNA-m5C regulatory atlas of human fetal tissues uncover the activities of Nsun2/Jarid2/Alyref axis

• High levels of m5C methylation exist on mRNA of various organ tissues, especially brain tissues during the development of human fetus from early to middle pregnancy. • m5C modification is positively correlated with gene expression homeostasis, which involved in regulating the stability of fetal development. • Nsun2 cKO and Nsun5 KO reduce neuronal proliferation, increase apoptosis, affect m5C levels, neuronal maturation and DNA repair. • Nsun2-mediated the m5C modification of Jarid2 affects axonogenesis and forebrain development via regulating H3K27me3 and chromatin accessibility, regulating. • Alyref could assist the influencing consequences of Nsun2 loss via recognizing m5C modification on Jarid2/Ezh2. Cytosine methylation (m5C) is a pivotal RNA modification essential for human fetal development, yet its tissue-specific distribution and regulatory mechanisms remain largely undefined. This study aimed to construct a tissue-specific atlas of m5C distribution across human fetal tissues and to dissect the epigenetic crosstalk between RNA methylation and epigenetic regulation. Specifically, we focused on how Nsun2-mediated m5C modifications influence chromatin dynamics and histone modification patterns during fetal development. We performed RNA bisulfite sequencing (m5C-Seq) and transcriptome profiling (RNA-Seq) across seven distinct human fetal tissues. To explore the functional roles of m5C, we employed Nsun2 conditional knockout and Nsun5 knockout mouse models. We further integrated multiple experimental approaches, including dot blot assays, immunofluorescence microscopy, Co-IP, CUT&Tag and ATAC-Seq, to examine the functional connections between m5C modification, histone modifications and chromatin accessibility. We established the first comprehensive atlas of m5C modifications across human fetal tissues, revealing distinct tissue-specific methylation patterns. We further demonstrated that m5C-modified transcripts maintain gene expression homeostasis and regulate critical developmental transcriptional programs through alternative splicing. Mechanistically, we found that Nsun2 recruits the Jarid2/Ezh2 complex and regulates its activity via m5C-dependent recognition by Alyref. This pathway coordinates histone modifications and chromatin accessibility, supporting proper fetal development. Our study presents a high-resolution, comprehensive tissue-specific m5C landscape during human fetal development, serving as a foundational resource for developmental epigenetics research. Subsequently, we identified a novel regulatory axis linking Nsun2, the Jarid2/Ezh2 complex, m5C modification patterns and Alyref functionality. This pathway integrates RNA methylation, histone modification and chromatin accessibility to precisely orchestrate fetal development, advancing our understanding of epigenetic regulation and offering new insights into potential therapeutic targets for developmental disorders.