NaBH4-sequencing of MVC infected WRD cells

RNA modifications play crucial roles in RNA metabolism, structure, and functions. N4-acetylcytidine (ac4C) modifications have been shown to enhance stability and translation efficiency of mRNAs and viral RNAs. However, the relationship between ac4C and alternative RNA processing remains unexplored. Here, N-acetyltransferase 10 (NAT10) and its catalyzed ac4C modifications on minute virus of canines (MVC) was shown to regulate viral DNA replication and RNA processing, including both the alternative RNA splicing and polyadenylation. Through acRIP-seq and RedaC:T-seq, functional ac4C-modified residue 3311 was identified and characterized, which affected MVC RNA processing, rather than altered the viral RNA stability. Ac4C modification at nt 3311 was revealed to participate in NP1-mediated viral RNA processing without influencing RNA affinity of NP1. Meanwhile, CPSF5 was identified to interact with NP1 and mediate viral RNA processing in an ac4C-dependent manner. Further in vitro assays showed that NP1 recruited CPSF5 to MVC RNAs and the ac4C modification promoted specific binding of CPSF5 to the target region, which ensured precise alternative MVC RNA processing. This study not only reveals the functions of NAT10 and ac4C, but also elucidates the mechanisms by which RNA modifications orchestrate MVC proteins and host factors for efficient viral replication and alternative RNA processing. The changes of ac4C modification in WRD cells after MVC infection were investigated by NaBH4 sequencing.