NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells

Abstract NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC–MS/MS) assays revealed noticeable ac4C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac4C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac4C writer enzyme, led to ac4C level decrease on target genes, promoted the core pluripotency regulator OCT4 (POU5F1) transcript decay, and finally impaired self-renewal and promoted early differentiation in hESCs. Together, our work presented here elucidates a previously unrecognized interconnectivity between the core pluripotent transcriptional network for the maintenance of human ESC self-renewal and NAT10-catalyzed ac4C RNA epigenetic modification. Graphical Abstract Graphical Abstract The core pluripotency regulator OCT4 functions as a key NAT10-responsive ac4C modification target during hESC self-renewal maintenance.