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. Overall design: 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, 0.2µl/sample) were added as spike-in for further calibration.