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

NAT10-catalyzed N-4-acetylcytidine (ac(4)C) 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 ac(4)C-targeted RNA immunoprecipitation sequencing (ac(4)C-RIP-seq), NaCNBH3-based chemical ac(4)C sequencing (ac(4)C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays revealed noticeable ac(4)C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac(4)C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac(4)C writer enzyme, led to ac(4)C 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 ac(4)C RNA epigenetic modification.

Automated summary

NAT10-catalyzed N-4-acetylcytidine (ac(4)C) plays an important role in post-transcriptional regulation of mRNA stability. This study shows that NAT10 expression is correlated with pluripotency and that ac(4)C modification preferentially targets core pluripotency transcription factors in hESCs. Inactivation of NAT10 leads to decreased ac(4)C levels, decay of core pluripotency regulator OCT4 transcript, and impairment of self-renewal and early differentiation in hESCs.