Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes
Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes
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DOI：
10.1074/jbc.m110.155523
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Yamashita, Masakane
中科院分区：
文献类型：
--
作者：
Ota, Ryoma;Kotani, Tomoya;Yamashita, Masakane
卵母细胞成熟的正常进展需要精确控制卵母细胞中储存的休眠 mRNA 的翻译激活时间。我们之前表明，Pumilio1 (Pum1) 在非洲爪蟾卵母细胞成熟过程中与细胞质多腺苷酸化元件结合蛋白 (CPEB) 合作，特异性参与细胞周期蛋白 B1 mRNA 的翻译控制。据报道，非洲爪蟾卵母细胞中存在另一种 Pumilio，Pumilio2 (Pum2)，该蛋白与无精子样删除蛋白 (DAZL) 一起调节 RINGO mRNA 的翻译。在这项研究中，我们通过使用新生产的区分 Pum1 和 Pum2 的抗体对 Pum1 和 Pum2 进行生化表征。 Pum1 和 Pum2 与未成熟卵母细胞中参与休眠 mRNA 翻译控制的几个关键蛋白结合，包括 CPEB 和 DAZL。然而，Pum1 和 Pum2 本身没有物理相互作用。注射抗 Pum1 或抗 Pum2 抗体可加速 CPEB 磷酸化、细胞周期蛋白 B1 翻译和卵母细胞成熟。 Pum1 磷酸化与 CPEB 从 Pum1 解离以及 Pum1 靶标细胞周期蛋白 B1 mRNA 的翻译激活同时发生，而 Pum2 磷酸化发生的时间早于 Pum1。一些（但不是全部）细胞周期蛋白 B1 mRNA 在卵母细胞成熟过程中释放去腺苷酸酶 PARN，而 Pum1 仍与 mRNA 相关。基于这些发现，我们讨论了 Pum1 和 Pum2 在卵母细胞成熟过程中 mRNA 翻译控制中的功能。
Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.