Abstract

Liquid–liquid phase separation (LLPS) underlies the formation of membraneless compartments in mammal cells. However, there are few reports that focus on the correlation of mouse oocyte maturation with LLPS. Previous studies have reported that paraspeckle component 1 (PSPC1) is related to the occurrence and development of tumors, but whether PSPC1 functions in mouse oocyte maturation is still unclear. Sequence analysis of PSPC1 protein showed that it contains a prion-like domain (PrLD) that is required for phase separation of proteins. In this study, we found that PSPC1 could undergo phase separation. Moreover, the loss of PrLD domain of PSPC1 could greatly weaken its phase separation ability. The immunofluorescence assays showed that PSPC1 is present in mouse oocytes in the germinal vesicle (GV) stage. Knockdown of PSPC1 significantly impeded the maturation of mouse oocytes in vitro. CHK1 has been reported to play important roles in the GV stage of mouse oocytes. Co-IP experiment revealed that PSPC1 could interact with phosphatase serine/threonine-protein phosphatase 5 (PPP5C), which regulates CHK1 phosphorylation. Western blot analysis revealed that PSPC1 could regulate the phosphorylation of CHK1 through PPP5C; however, PSPC1 without PrLD domain was inactive, suggesting that the lack of phase separation ability led to the abnormal function of PSPC1 in regulating CHK1 phosphorylation. Thus, we conclude that PSPC1 may undergo phase separation to regulate the phosphorylation level of CHK1 via PPP5C and participate in mouse oocyte maturation. Our study provides new insights into the mechanism of mouse oocyte maturation.

中文翻译：

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PSPC1通过相分离调节CHK1磷酸化并参与小鼠卵母细胞成熟

摘要

液-液相分离 (LLPS) 是哺乳动物细胞中无膜隔室形成的基础。然而，很少有报道关注小鼠卵母细胞成熟与 LLPS 的相关性。既往研究报道，paraspeckle component 1 (PSPC1)与肿瘤的发生、发展有关，但PSPC1是否在小鼠卵母细胞成熟中发挥作用尚不清楚。PSPC1 蛋白的序列分析表明，它包含蛋白质相分离所需的朊病毒样结构域 (PrLD)。在这项研究中，我们发现 PSPC1 可以发生相分离。此外，PSPC1 的 PrLD 结构域的丢失会大大削弱其相分离能力。免疫荧光测定表明，PSPC1 存在于生泡 (GV) 阶段的小鼠卵母细胞中。PSPC1的击倒显着阻碍了小鼠体外卵母细胞的成熟。据报道，CHK1 在小鼠卵母细胞的 GV 阶段发挥重要作用。Co-IP 实验表明，PSPC1 可以与调节 CHK1 磷酸化的磷酸酶丝氨酸/苏氨酸蛋白磷酸酶 5 (PPP5C) 相互作用。Western blot分析显示PSPC1可以通过PPP5C调控CHK1的磷酸化；然而，没有PrLD结构域的PSPC1是不活跃的，这表明缺乏相分离能力导致PSPC1在调节CHK1磷酸化方面的功能异常。因此，我们得出结论，PSPC1可能经历相分离以通过PPP5C调节CHK1的磷酸化水平并参与小鼠卵母细胞的成熟。我们的研究为小鼠卵母细胞成熟的机制提供了新的见解。