NAT10-mediated mRNA N4-acetylation is Essential for the Translational Regulation During Oocyte Meiotic Maturation in Mice

Mammalian oocyte maturation is driven by strictly translational regulation of maternal mRNAs stored in the cytoplasm. However, the function and mechanism of post-transcriptional chemical modifications especially the newly identified N4-acetylcytidine (ac4C) catalyzed by N-acetyltransferase 10 (NAT10) in this process are previously unknown. In this study, we developed a low-input ac4C sequencing technology--ac4C LACE-seq and mapped 8241 ac4C peaks at the whole transcriptome level using 50 mouse oocytes at the germinal vesicle (GV) stage. We profiled the mRNA landscapes of NAT10-interactions and ac4C modifications. The NAT10-interacted and ac4C modified transcripts displayed association with high translation efficiency in oocytes. Oocyte-specific Nat10 knockout wiped out ac4C signals in oocytes and caused severe defects in meiotic maturation and female infertility. ac4C LACE-seq results indicated that Nat10 deletion led to a failure of ac4C deposition on mRNAs encoding key maternal factors such as MAY2, ZAR1, BTG4 and cyclin B1 that regulate transcriptome stability and maternal-to-zygotic transition. Nat10-deleted oocytes had decreased mRNA translation efficiencies during meiotic maturation, partially due to the direct inhibition ac4C sites on specific transcripts. In sum, we developed low-input, high-sensitivity mRNA ac4C profiling approach and highlighted the important physiological function of ac4C in precise regulation of the oocyte meiotic maturation by enhancing translation efficiency. The oocytes were harvested from mature WT and Gdf9-cre Nat10 conditional knockout female mice 32-48h after PMSG and washed by 0.2% containing PBS for 3 times, then 10 oocytes per sample were lysed in lysis buffer (containing 0.2% Triton X-100 and Recombination RNase Inhibitor) and prceeded for cDNA library constribution following the workflow of Smart-seq2. And for each sample, ERCC (1:1000, 0.2ul/sample) were added as spike-in for further calibration.哺乳动物卵母细胞成熟过程，由储存在细胞质中的母体信使RNA（mRNA）的严格翻译调控所驱动。然而，此前对于该过程中发生的转录后化学修饰，尤其是由N-乙酰基转移酶10（N-acetyltransferase 10, NAT10）催化的新发现的N4-乙酰胞苷（N4-acetylcytidine, ac4C）的功能与作用机制仍不明晰。本研究中，我们开发了一种低起始量ac4C测序技术——ac4C LACE-seq，并利用50枚处于生发泡（germinal vesicle, GV）期的小鼠卵母细胞，在全转录组层面绘制了8241个ac4C修饰峰。我们对NAT10结合转录本以及ac4C修饰的mRNA图谱进行了表征。NAT10结合并经ac4C修饰的转录本，与卵母细胞内的高翻译效率呈现显著关联。卵母细胞特异性敲除Nat10可完全消除卵母细胞中的ac4C修饰信号，并导致减数分裂成熟严重缺陷以及雌性不育。ac4C LACE-seq结果显示，敲除Nat10会导致编码关键母体因子（如MAY2、ZAR1、BTG4以及细胞周期蛋白B1）的mRNA无法发生ac4C修饰沉积，而这些因子可调控转录组稳定性以及母源-合子转换过程。敲除Nat10的卵母细胞在减数分裂成熟期间，mRNA翻译效率有所降低，这在一定程度上源于特定转录本上ac4C位点的直接调控作用。综上，我们开发了一种低起始量、高灵敏度的mRNA ac4C图谱分析方法，并揭示了ac4C通过提升翻译效率，精准调控卵母细胞减数分裂成熟的重要生理功能。本研究中的卵母细胞取自经孕马血清促性腺激素（PMSG）处理32-48小时后的成熟野生型（wild type, WT）以及Gdf9-cre介导的Nat10条件性敲除雌性小鼠；随后将卵母细胞用含0.2%组分的磷酸盐缓冲液（PBS）洗涤3次，随后每份样本取10枚卵母细胞，在含有0.2% Triton X-100与重组RNase抑制剂的裂解缓冲液中进行裂解，并按照Smart-seq2的实验流程构建cDNA文库。此外，每份样本中均加入1:1000稀释的ERCC（0.2μl/样本）作为外源内参，用于后续校准。