PCBP1/2 and TDP43 Function as NAT10 Adaptors to Mediate mRNA ac⁴C Formation in Mammalian Cells

Massive numbers of modified bases in mRNAs sculpt the epitranscriptome and play vital roles in RNA metabolism. The only known acetylated RNA modification, N-4-acetylcytidine (ac⁴C), is highly conserved across cell types and among species. Although the GCN5-related acetyltransferase 10 (NAT10) functions as an ac⁴C writer, the mechanism underlying the acetylation process is largely unknown. In this study, the NAT10/PCBP/TDP43 complex mediated mRNA ac⁴C formation in mammalian cells is identified. RNA-binding proteins (RBPs) are identified, affiliated with two different families, poly(rC)-binding protein 1/2 (PCBP1/2) and TAR DNA binding protein 43 (TDP43), as NAT10 adaptors for mRNA tethering and substrate selection. Knockdown of the adaptors resulted in decreased mRNA acetylation abundance in HEK293T cells and ablated cytidine-rich ac⁴C motifs. The adaptors also affect the ac⁴C sites by recruiting NAT10 to their binding sequences. The presence of the NAT10/PCBP/TDP43 complex in mouse testes highlights its potential physiological functions in vivo. These findings reveal the composition of the mRNA ac⁴C writer complex in mammalian cells and expand the knowledge of mRNA acetylation and ac⁴C site preferences.