Saitoh Atsushi, Takada Yuki, Horie Mayu, Kotani Tomoya
1Biosystems Science Course,Graduate School of Life Science,Hokkaido University,Sapporo 060-0810,Japan.
Zygote. 2018 Oct;26(5):372-380. doi: 10.1017/S0967199418000369. Epub 2018 Oct 5.
SummaryTranslational regulation of mRNAs is crucial for promoting various cellular and developmental processes. Pumilio1 (Pum1) has been shown to play key roles in translational regulation of target mRNAs in many systems of diverse organisms. In zebrafish immature oocytes, Pum1 was shown to bind to cyclin B1 mRNA and promote the formation of cyclin B1 RNA granules. This Pum1-mediated RNA granule formation seemed critical to determine the timing of translational activation of cyclin B1 mRNA during oocyte maturation, leading to activation of maturation/M-phase-promoting factor (MPF) at the appropriate timing. Despite its fundamental importance, the mechanisms of translational regulation by Pum1 remain elusive. In this study, we examined the phosphorylation of Pum1 as a first step to understand the mechanisms of Pum1-mediated translation. SDS-PAGE analyses and phosphatase treatments showed that Pum1 was phosphorylated at multiple sites during oocyte maturation. This phosphorylation began in an early period after induction of oocyte maturation, which preceded the polyadenylation of cyclin B1 mRNA. Interestingly, depolymerization of actin filaments in immature oocytes caused phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and polyadenylation of cyclin B1 mRNA but not translational activation of the mRNA. Overexpression of the Pum1 N-terminus prevented the phosphorylation of Pum1, disassembly of cyclin B1 RNA granules, and translational activation of the mRNA even after induction of oocyte maturation. These results suggest that Pum1 phosphorylation in the early period of oocyte maturation is one of the key processes for promoting the disassembly of cyclin B1 RNA granules and translational activation of target mRNA.
摘要
mRNA的翻译调控对于促进各种细胞和发育过程至关重要。在多种生物的许多系统中，Pumilio1（Pum1）已被证明在靶mRNA的翻译调控中起关键作用。在斑马鱼未成熟卵母细胞中，Pum1被证明与细胞周期蛋白B1 mRNA结合并促进细胞周期蛋白B1 RNA颗粒的形成。这种由Pum1介导的RNA颗粒形成似乎对于确定卵母细胞成熟过程中细胞周期蛋白B1 mRNA翻译激活的时间至关重要，从而在适当的时间激活成熟/促有丝分裂因子（MPF）。尽管其具有根本重要性，但Pum1介导的翻译调控机制仍然难以捉摸。在本研究中，我们研究了Pum1的磷酸化作为理解Pum1介导的翻译机制的第一步。SDS-PAGE分析和磷酸酶处理表明，Pum1在卵母细胞成熟过程中在多个位点被磷酸化。这种磷酸化在卵母细胞成熟诱导后的早期开始，早于细胞周期蛋白B1 mRNA的多聚腺苷酸化。有趣的是，未成熟卵母细胞中肌动蛋白丝的解聚导致Pum1的磷酸化、细胞周期蛋白B1 RNA颗粒的解体和细胞周期蛋白B1 mRNA的多聚腺苷酸化，但不会导致该mRNA的翻译激活。即使在诱导卵母细胞成熟后，Pum1 N端的过表达也能阻止Pum1的磷酸化、细胞周期蛋白B1 RNA颗粒的解体和mRNA的翻译激活。这些结果表明，卵母细胞成熟早期的Pum1磷酸化是促进细胞周期蛋白B1 RNA颗粒解体和靶mRNA翻译激活的关键过程之一。
