Biochemical identification of Xenopus pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein
Biochemical identification of Xenopus pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein
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DOI：
10.1074/jbc.m010528200
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发表时间：
2001-06-15
影响因子：
4.8
通讯作者：
Yamashita, M
中科院分区：
文献类型：
--
作者：
Nakahata, S;Katsu, Y;Yamashita, M
储存在卵母细胞中的休眠细胞周期蛋白B1 mRNA的翻译激活是许多脊椎动物卵母细胞成熟的启动或促进的先决条件。使用抗与果蝇Pumilio高度同源的结构域的单克隆抗体，我们首次在任何脊椎动物中表明Pumilio的同源物在爪蟾卵母细胞中表达，该137-kDa蛋白质结合到包括细胞周期蛋白B1 mRNA的3 ′-非翻译区的核苷酸1335-1338处的序列UGUA的区域，其接近但不与胞质多聚腺苷酸化元件（CPE）重叠。通过蛋白质下拉试验证明了爪蟾Pumilio与爪蟾Nanos同系物（Xcat-2）的体外物理关联。免疫沉淀实验结果表明，在卵母细胞中，CPE结合蛋白（CPEB）与非洲爪蟾之间存在相互作用，CPEB是卵母细胞中转录抑制和激活mRNA的关键调节因子，这一结果为进一步研究卵母细胞成熟过程中mRNA 3 '端的转录调控机制提供了新的线索。这些结果也表明了Pumilio作为无脊椎动物和脊椎动物中休眠mRNA的翻译调节剂的功能的普遍性。
Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes, This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stared in oocytes, This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.