Poly(A) polymerase and the regulation of cytoplasmic polyadenylation
Poly(A) polymerase and the regulation of cytoplasmic polyadenylation
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DOI：
10.1074/jbc.m103030200
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发表时间：
2001-11-09
影响因子：
4.8
通讯作者：
Wickens, M
中科院分区：
文献类型：
--
作者：
Dickson, KS;Thompson, SR;Wickens, M
卵母细胞和胚胎中的翻译激活通常通过增加聚腺苷酸长度来调节。切割和聚腺苷酸化特异性因子 (CPSF)、细胞质聚腺苷酸化元件结合蛋白 (CPEB) 和聚腺苷酸聚合酶 (PAP) 均与非洲爪蟾卵母细胞的细胞质聚腺苷酸化有关。细胞质多腺苷酸化活性首先出现在脊椎动物卵母细胞减数分裂成熟过程中。这里提供的数据表明，在减数分裂成熟之前或之后制备的非洲虎卵母细胞提取物中存在含有 CPSF 和 CPEB 的复合物。对作为聚腺苷酸化底物的多种 RNA 序列的评估表明，鸡蛋提取物中聚腺苷酸化的序列特异性与用高度纯化的哺乳动物 CPSF 和重组 PAP 观察到的相当。通过将相同的 RNA 注射到卵母细胞中进行评估，这两个体外系统表现出与体内观察到的相似但不相同的序列特异性。这些发现意味着 CPSF 内在的 RNA 序列偏好足以解释某些 mRNA 细胞质多腺苷酸化的特异性。我们讨论了所有聚腺苷酸化反应都需要 CPSF 的假设，但某些 mRNA 的聚腺苷酸化可能需要其他因素，例如 CPEB。为了测试体内 PAP 与 mRNA 结合的结果，使用 MS2 外壳蛋白将 PAP 与静息卵母细胞中的报告基因 mRNA 结合。束缚的 PAP 催化聚腺苷酸化并刺激翻译，类似于 40 倍；刺激完全是顺式作用，但独立于 CPE 和 AAUAAA。聚腺苷酸化和翻译刺激都需要 PAP 催化核心，但不需要 PAP 的假定 CPSF 相互作用结构域。这些结果表明，PAP 的过早募集会导致静息卵母细胞中早熟的多聚腺苷酸化和翻译刺激，并且可以解释为其他因素的作用是将 PAP 传递到 mRNA。
Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation similar to 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.