Zhang Xueyan, Qin Shuangkang, Huang Fang, Liu Haizhou, Wang Jun, Chen Zhen, Hao Haojie, Ding Shuang, Liu Lishi, Yu Baocheng, Liu Yi, Liu Haibin, Guan Wuxiang
Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China.
Hubei Jiangxia Laboratory, Wuhan, Hubei 430200, China.
Nucleic Acids Res. 2025 Mar 20;53(6). doi: 10.1093/nar/gkaf229.
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 messenger RNAs 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) were 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.
RNA修饰在RNA代谢、结构和功能中发挥着关键作用。N4-乙酰胞苷（ac4C）修饰已被证明可增强信使RNA和病毒RNA的稳定性及翻译效率。然而，ac4C与可变RNA加工之间的关系仍未得到探索。在此，研究表明N-乙酰转移酶10（NAT10）及其催化的犬微小病毒（MVC）上的ac4C修饰可调节病毒DNA复制和RNA加工，包括可变RNA剪接和多聚腺苷酸化。通过acRIP-seq和RedaC:T-seq，鉴定并表征了功能性ac4C修饰的3311位残基，其影响MVC RNA加工而非改变病毒RNA稳定性。研究发现3311位核苷酸处的ac4C修饰参与NP1介导的病毒RNA加工，而不影响NP1与RNA的亲和力。同时，鉴定出CPSF5与NP1相互作用，并以ac4C依赖的方式介导病毒RNA加工。进一步的体外实验表明，NP1将CPSF5招募至MVC RNA，ac4C修饰促进CPSF5与靶区域的特异性结合，从而确保精确的MVC可变RNA加工。本研究不仅揭示了NAT10和ac4C的功能，还阐明了RNA修饰协调MVC蛋白和宿主因子以实现高效病毒复制和可变RNA加工的机制。
