NSUN5 is essential for proper Hippo signaling in mouse preimplantation development

AbstractNOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. By re-analyzing published RNA-sequencing data, we found that Nsun5 expression was increased at the 2-cell stage during mouse preimplantation development. It has been reported that m5C regulates the stability of rRNA and mRNA. Therefore, NSUN5 may affect the expression of genes required for the development and differentiation of early mouse embryos via its role in modifying RNA. The Hippo signaling pathway has been identified as a critical regulator of the segregation of inner cell mass and trophectoderm lineages during mouse embryogenesis. According to these findings, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. Using Nsun5-specific small interfering RNAs, we efficiently knocked down Nsun5 expression in mouse preimplantation embryos, resulting in impairments in early development, including reduced blastocyst formation, smaller size, and impaired hatching from the zona pellucida. Nsun5 depletion also led to decreased cell numbers, suggesting impaired cell proliferation. Furthermore, Nsun5 knockdown embryos exhibited reduced yes-associated protein 1 (YAP1) nuclear translocation during the morula stage, potentially affecting cell differentiation. The imbalance in pluripotent and trophectoderm lineages was evident with a significant reduction in the ratio of CDX2-positive cells to OCT4-positive cells. Nsun5 depletion impacted the Hippo signaling pathway, increasing the expression of the key genes Lats1 and Lats2 during the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway. Understanding the roles of Nsun5 in mouse early embryos will provide insights into complex regulatory networks in development, with implications for reproductive medicine and fertility research.