An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro
An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro
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DOI：
10.1128/mcb.19.7.4552
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发表时间：
1999-07
影响因子：
5.3
通讯作者：
Zuo-ren Wang;N. Day;P. Trifillis;M. Kiledjian
中科院分区：
文献类型：
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作者：
Zuo-ren Wang;N. Day;P. Trifillis;M. Kiledjian
稳定的珠蛋白mRNA为研究哺乳动物mRNA更新机制提供了理想的系统。α-珠蛋白mRNA稳定性由3′非翻译区（3′UTR）中的序列决定，该序列形成特异性核糖核蛋白复合物（α-复合物），其存在与mRNA稳定性相关。该复合物中的主要蛋白质组分之一是两个多胞苷酸结合蛋白α CP 1和α CP 2家族。利用体外转录和多聚腺苷酸化的α-珠蛋白3′UTR，我们设计了一种体外mRNA衰变试验，该试验重现了在细胞中观察到的α-复合物依赖性mRNA稳定性。将RNA与红白血病K562胞质提取物一起孵育，导致具有含有约30个核苷酸的周期性的不同中间体的去腺苷酸化，这与多聚（A）结合蛋白（PABP）单体的结合一致。通过螯合α CP 1和α CP 2破坏α-复合物可增强mRNA的去腺苷化和降解，而α-复合物的重建可稳定mRNA。类似地，PABP也是mRNA在体外的稳定性所必需的，因为在其耗尽后导致快速去腺苷化。α CP 1和α CP 2与PABP之间的相互作用依赖于RNA，表明α-复合物可以直接与PABP相互作用。因此，α-复合物是一种体外mRNA稳定性复合物，至少部分通过与PABP相互作用发挥功能。
ABSTRACT The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. α-Globin mRNA stability is dictated by sequences in the 3′ untranslated region (3′UTR) which form a specific ribonucleoprotein complex (α-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, αCP1 and αCP2. Using an in vitro-transcribed and polyadenylated α-globin 3′UTR, we have devised an in vitro mRNA decay assay which reproduces the α-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the α-complex by sequestration of αCP1 and αCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the α-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between αCP1 and αCP2 with PABP suggests that the α-complex can directly interact with PABP. Therefore, the α-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.