NAT10-mediated mRNA N4-acetylcytidine is Essential for the Translational Regulation of 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. We developed a low-input ac4C sequencing technology - ac4C LACE-seq and mapped 8241 ac4C peaks at the whole transcriptome level using 50 mouse GV oocyte. In order to study the impact of Nat10 deletion on ac4C modification, we used ac4C LACE-seq technology to detect changes in ac4C modification in wild type and Nat10-GKO.

哺乳动物卵母细胞成熟过程依赖于细胞质中储存的母源mRNA的严格翻译调控。然而，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）——在该过程中的功能与作用机制此前尚不明晰。本研究开发了低起始量ac4C测序技术——ac4C LACE-seq，并利用50枚生发泡（germinal vesicle, GV）期小鼠卵母细胞，在全转录组范围内定位到8241个ac4C修饰峰。我们绘制了NAT10结合与ac4C修饰的mRNA全景图谱。结合NAT10且带有ac4C修饰的转录本，在卵母细胞中呈现出较高的翻译效率。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与雌性不育。ac4C LACE-seq结果显示，Nat10缺失会导致关键母源因子编码mRNA上的ac4C修饰沉积失败，这些母源因子包括MAY2、ZAR1、BTG4以及细胞周期蛋白B1（cyclin B1），它们参与调控转录组稳定性与母源-合子转换（maternal-to-zygotic transition）。Nat10缺失的卵母细胞在减数分裂成熟过程中mRNA翻译效率下降，这在一定程度上源于特定转录本上的ac4C位点所介导的直接调控受阻。综上，本研究开发了低起始量、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。本研究再次开发了低起始量ac4C测序技术——ac4C LACE-seq，并利用50枚小鼠GV期卵母细胞完成了全转录组水平的8241个ac4C峰定位。为探究Nat10缺失对ac4C修饰的影响，我们采用ac4C LACE-seq技术检测了野生型与Nat10-GKO（卵母细胞特异性Nat10敲除）小鼠卵母细胞中的ac4C修饰变化。