Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as ‘Trim away’ (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

中文翻译：

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COP9 信号体亚基 3 通过在小鼠卵母细胞中形成稳定的蛋白质沉积，对于早期胚胎的存活是必需的


对早期哺乳动物发育所需基因的研究因母体产物的蛋白质沉积而变得复杂，这些蛋白质沉积在基因位点被破坏后仍继续发挥作用。这导致表型表现延迟，并低估细胞全能性胚胎期所需的已知基因数量。在这里，我们通过证明 Cops3 基因在小鼠发育的 2 细胞阶段保护基因组完整性来揭示基因 Cops3 的关键作用，这与之前植入后的功能分配形成鲜明对比。这种新的作用是由大量蛋白质沉积（蛋白质组的第 94 个百分位）介导的，蛋白质沉积分为异常稳定的皮质边缘（卵母细胞中普遍存在）和胚胎核中的辅助沉积。由于蛋白质丰度和稳定性阻碍了卵母细胞中基于 DNA 或 RNA 的基因失活的前景，因此我们利用了一种经典方法以及一种新兴的蛋白质失活方法：抗原掩蔽（用于功能抑制）与 TRIM21 介导的蛋白酶体降解（也称为“修剪掉”（物理去除）。与接受抗绿色荧光蛋白的胚胎不同，两者都会导致双细胞胚胎死亡。 COPS3蛋白靶向和非靶向胚胎之间的比较揭示了大规模的转录组差异，这对于与RNA代谢和保存基因组完整性至关重要的生物功能相关的基因最为明显。通过 2 细胞胚胎中 DNA 内复制和 DNA 链断裂的发生，原位证实了与 COPS3 失活相关的基因表达异常。这些结果将 Cops3 招募到早期胚胎存活所必需的基因小家族中。 总体而言，如果这些基因的蛋白质产物在卵母细胞中积累，那么指定在胚胎发生中发挥作用的基因可能不如假设的安全：蛋白质水平上的基因失活可能会暴露出比常规基因等遗传技术所识别的更早的表型沉默。