EXOSC10 sculpts the transcriptome during oocyte growth-to-maturation transition. EXOSC10 sculpts the transcriptome during oocyte growth-to-maturation transition

Growing mammalian oocytes accumulate substantial amounts of RNA, most of which are degraded during the subsequent maturation stage. The growth-to-maturation transition begins with germinal vesicle breakdown (GVBD, envisioned as nuclear envelope breakdown) and is critical for oocyte quality. However, the concomitant changes in the transcriptome during GVBD as well as the underlying machinery remained unclear. Here, we report that an RNA exosome-associated RNase, EXOSC10, sculpts the transcriptome at multiple level to facilitate the oocyte growth-to-maturation transition. We establish an oocyte-specific knockout of Exosc10 in mice using CRISPR/Cas9 and find female subfertility due to failed GVBD. By performing single oocyte RNA-seq in different ways, we document dysregulated transcriptomes, unsuccessfully processed rRNAs in mutant oocytes, and many up-regulated RNAs that encode proteins important for endomembrane trafficking, meiotic cell cycle and RNA metabolism. EXOSC10-depleted oocytes have impaired endomembrane components including endosome, lysosome, ER and Golgi. In addition, CDK1 fails to be activated possibly due to persistent WEE1 activity, which blocked lamina phosphorylation and disassembly in mutant oocytes. Collectively, we propose that EXOSC10 promotes the growth-to-maturation transition in mouse oocytes by degrading mRNAs that encode growth-phase factors and sculpting the transcriptome to support the maturation phase of oogenesis. Overall design: There are 64 single oocyte polyA-RNA libraries generated with poly(A)-based enrichment and reverse transcription, including 22 at GV stage, 19 at GV+3hr stage, and 23 at MII stage. In addition, there are 22 single single oocyte RiboMinus libraries generated independent of poly(A), including 15 at GV stage and 7 at MII stage. Morever, there are 22 single oocyte rRNA libraries generated by total RNA extraction without ribosome depletion. Each stage contains control oocytes and Exosc10 oocyte-specific knockout oocytes (cKO). The information of the stage, genotype, female ID and oocyte ID is in the name of each file: stage_genotype_femaleID_oocyteID. In each oocyte, equal amounts of ERCC spike-in mix was added.

生长中的哺乳动物卵母细胞会积累大量RNA，其中绝大多数会在后续的成熟阶段被降解。卵母细胞从生长到成熟的转换以生发泡破裂（germinal vesicle breakdown, GVBD）为起始，这一过程对卵母细胞质量至关重要。然而，GVBD过程中转录组的伴随变化及其潜在调控机制仍未明确。本研究发现，一种与RNA外泌体相关的核糖核酸酶EXOSC10可通过多层面重塑转录组，以促进卵母细胞生长向成熟的转换过程。我们利用CRISPR/Cas9技术构建了小鼠卵母细胞特异性Exosc10敲除模型，发现敲除小鼠因GVBD失败而出现雌性生育力下降。通过开展多种方式的单卵母细胞RNA测序，我们揭示了突变型卵母细胞中转录组失调、核糖体RNA（rRNA）加工异常，以及大量编码参与内膜运输、减数分裂细胞周期和RNA代谢相关蛋白的mRNA表达上调。敲低EXOSC10的卵母细胞中，包括内体、溶酶体、内质网（ER）和高尔基体在内的内膜系统组分均出现功能受损。此外，由于WEE1活性持续存在，细胞周期蛋白依赖性激酶1（CDK1）无法被激活，这阻碍了突变型卵母细胞中的核纤层磷酸化与解聚。综上，本研究提出EXOSC10可通过降解编码生长阶段因子的mRNA，并重塑转录组以支持卵发生的成熟阶段，从而促进小鼠卵母细胞的生长向成熟转换。

实验设计：本研究共构建64个基于poly(A)富集与反转录的单卵母细胞polyA-RNA测序文库，其中包括22个GV期、19个GV+3小时期以及23个MII期的文库。此外，本研究还构建了22个不依赖poly(A)的单卵母细胞RiboMinus测序文库，其中15个为GV期，7个为MII期。另外，本研究还通过总RNA提取且未进行核糖体去除的方式，构建了22个单卵母细胞rRNA测序文库。每个时期的样本均包含野生型对照卵母细胞与Exosc10卵母细胞特异性敲除（cKO）卵母细胞。每个样本文件的命名中包含了样本时期、基因型、雌性个体ID以及卵母细胞ID，命名格式为：stage_genotype_femaleID_oocyteID。每个卵母细胞样本中均加入了等量的ERCC外参混合液（ERCC spike-in mix）。