NaBH4-sequencing of MVC infected WRD cells. 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. Overall design: The changes of ac4C modification in WRD cells after MVC infection were investigated by NaBH4 sequencing.

RNA修饰在RNA代谢、结构与功能中发挥关键作用。N4-乙酰胞苷（N4-acetylcytidine，ac4C）修饰已被证实可增强mRNA与病毒RNA的稳定性及翻译效率。然而，ac4C与可变RNA加工之间的关联仍未被探明。本研究证实，N-乙酰转移酶10（N-acetyltransferase 10，NAT10）及其催化的犬微小病毒（minute virus of canines，MVC）基因组上的ac4C修饰，可调控病毒DNA复制与RNA加工过程，涵盖可变RNA剪接与多聚腺苷酸化两个环节。本研究通过acRIP-seq与RedaC:T-seq技术，鉴定并表征了具有功能活性的ac4C修饰残基3311，该位点仅影响MVC的RNA加工过程，并未改变病毒RNA的稳定性。研究发现，位于核苷酸3311（nt 3311）位点的ac4C修饰可参与NP1介导的病毒RNA加工过程，且不会影响NP1的RNA结合亲和力。与此同时，本研究鉴定到切割与多聚腺苷酸化特异性因子5（CPSF5）可与NP1相互作用，并以ac4C依赖的方式介导病毒RNA加工过程。进一步的体外实验表明，NP1可将CPSF5招募至MVC RNA上，而ac4C修饰可促进CPSF5与靶区域的特异性结合，从而保障MVC可变RNA加工的精准性。本研究不仅揭示了NAT10与ac4C的功能，还阐明了RNA修饰通过协调调控MVC蛋白与宿主因子，实现高效病毒复制与可变RNA加工的分子机制。整体实验设计：本研究采用NaBH4测序技术，探究了MVC感染后WRD细胞中ac4C修饰的水平变化。