Data_Sheet_1_NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.xlsx

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

N4-乙酰胞嘧啶核苷（N4-acetylcytidine，ac4C）是新近发现的信使RNA（mRNA）表观遗传修饰，已被证实为调控mRNA稳定性与翻译效率的关键因子。然而，ac4C在卵母细胞成熟过程中的作用尚未被探究——该过程主要由转录后调控机制主导。N-乙酰基转移酶10（N-acetyltransferase 10，NAT10）是目前已知的哺乳动物体内唯一催化ac4C生成的酶，且迄今尚无ac4C结合蛋白的相关报道。本研究中，我们观察到从小鼠未成熟卵母细胞到成熟卵母细胞的进程中，ac4C水平与NAT10的表达均呈下降趋势。通过小干扰RNA（siRNA）介导生发泡（GV）期卵母细胞的NAT10敲低后，ac4C修饰水平显著降低，体外减数分裂成熟过程亦明显受阻。具体而言，与未转染的对照卵母细胞（74.6%）及转染阴性对照siRNA的卵母细胞（72.6%）相比，NAT10敲低组的第一极体排出率显著降至34.6%（p < 0.001）；而两组的生发泡破裂（GVBD）率则无显著差异（p = 0.6531）。通过对HEK293T细胞开展RNA免疫沉淀联合高通量测序分析，我们发现差异调控基因富集于核小体组装、染色质沉默、染色质修饰及细胞骨架锚定等生物学过程。此外，我们借助生物信息学算法结合HEK293T细胞的RNA下拉实验，鉴定出TBL3为潜在的ac4C结合蛋白，其可能介导下游细胞活动。综上，本研究结果表明，NAT10介导的ac4C修饰是体外卵母细胞成熟过程中的重要调控因子，且TBL3是潜在的ac4C结合蛋白。