Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.
Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.
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DOI：
10.1101/gad.5.9.1697
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发表时间：
1991-09
影响因子：
10.5
通讯作者：
J. Paris;K. Swenson;H. Piwnica-Worms;J. Richter
中科院分区：
文献类型：
--
作者：
J. Paris;K. Swenson;H. Piwnica-Worms;J. Richter
在非洲爪蟾卵母细胞成熟过程中，poly（A）延伸控制具有CPE（细胞质聚腺苷酸化元件）的特定mRNA的翻译募集。为了研究多聚腺苷酸化的激活，我们使用了通常不能进行多聚腺苷酸化的卵母细胞提取物。添加含有杆状病毒表达的细胞周期蛋白的细胞裂解物到这些提取物中诱导外源性B4 RNA的聚腺苷酸化。p34 cdc 2激酶参与细胞周期蛋白介导的多聚腺苷酸化被证明由p13-琼脂糖耗尽;去除从卵母细胞提取物与这种亲和基质的激酶废除多聚腺苷酸化激活。然而，重新引入含有杆状病毒表达的p34 cdc 2的细胞裂解物完全恢复了这种活性。为了确定可能负责激活的多聚腺苷酸化装置的因素，我们采用UV交联，并确定了一个58 kD的蛋白质，结合卵母细胞提取物中的B4 CPE。在多聚腺苷酸化精通鸡蛋提取物，这种蛋白质有一个较慢的电泳迁移率，这表明翻译后修饰。一个类似的蛋白质的大小变化是显而易见的，在卵母细胞提取物补充裂解物含有杆状病毒表达的细胞周期蛋白和p34 cdc 2。这种大小的转变，这是逆转治疗与酸性磷酸酶，符合时间与细胞周期蛋白诱导的多聚腺苷酸化激活。我们建议，p34 cdc 2激酶活性导致磷酸化的58 kD CPE结合蛋白，这一事件是至关重要的细胞质聚腺苷酸化，发生在卵母细胞成熟。
During Xenopus oocyte maturation, poly(A) elongation controls the translational recruitment of specific mRNAs that possess a CPE (cytoplasmic polyadenylation element). To investigate the activation of polyadenylation, we have employed oocyte extracts that are not normally competent for polyadenylation. Addition of cell lysates containing baculovirus-expressed cyclin to these extracts induces the polyadenylation of exogenous B4 RNA. The involvement of p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, however, completely restores this activity. To identify factors of the polyadenylation apparatus that might be responsible for the activation, we employed UV cross-linking and identified a 58-kD protein that binds the B4 CPE in oocyte extracts. In polyadenylation-proficient egg extracts, this protein has a slower electrophoretic mobility, which suggests a post-translational modification. A similar size shift of the protein is evident in oocyte extracts supplemented with lysates containing baculovirus-expressed cyclin and p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34cdc2 kinase activity leads to the phosphorylation of the 58-kD CPE-binding protein and that this event is crucial for the cytoplasmic polyadenylation that occurs during oocyte maturation.