NSUN2-Mediated m⁵C Methylation Impairs Endometrial Receptivity

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m⁵C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m⁵C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m⁵C sites and genes was elucidated through m⁵C-BS-seq, whereas the overall m⁵C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m⁵C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets' expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9⁺ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2^OE) in the Ishikawa cell line elevated m⁵C levels and promoted cell proliferation and autophagy. NSUN2^OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m⁵C modification, and NSUN2^OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m⁵C and RIF and suggested a potential new therapeutic strategy for RIF.