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. 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，其中大部分会在后续的成熟阶段被降解。卵母细胞的生长向成熟转变始于生发泡破裂（germinal vesicle breakdown, GVBD，即核膜破裂过程），这一过程对卵母细胞质量至关重要。然而，GVBD期间转录组（transcriptome）的伴随变化及其潜在调控机制仍不明确。本研究发现，RNA外切体相关核糖核酸酶（ribonuclease, RNase）EXOSC10可通过多维度重塑转录组，以促进卵母细胞的生长向成熟转变。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，发现雌性小鼠因GVBD失败而出现生育力低下。通过多种方式开展单细胞卵母细胞RNA测序，我们发现突变型卵母细胞存在转录组失调、核糖体RNA（ribosomal RNA, rRNA）加工异常，以及大量编码与内膜运输、减数分裂细胞周期及RNA代谢相关蛋白的RNA发生上调。EXOSC10缺失的卵母细胞其内膜系统组分受损，包括内体、溶酶体、内质网（endoplasmic reticulum, ER）和高尔基体。此外，由于WEE1活性持续维持，细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1, CDK1）无法被激活，进而阻断了突变型卵母细胞的核纤层磷酸化与解聚过程。综上，我们提出EXOSC10可通过降解编码生长阶段因子的信使RNA（messenger RNA, 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变。 本研究共构建了64个基于聚腺苷酸（poly(A)）富集与反转录技术的单细胞卵母细胞polyA-RNA文库，其中GV期22个、GV+3小时期19个、MII期23个。此外，构建了22个不依赖poly(A)的单细胞卵母细胞RiboMinus文库，其中GV期15个、MII期7个。另有22个通过总RNA提取且未进行核糖体去除的单细胞卵母细胞rRNA文库。每个阶段均设置野生型对照卵母细胞与Exosc10卵母细胞特异性敲除（conditional knockout, cKO）卵母细胞。样本的阶段、基因型、雌性个体ID与卵母细胞ID信息包含在每个文件的命名中，命名格式为：stage_genotype_femaleID_oocyteID。每个卵母细胞中均加入了等量的ERCC外源对照混合液。