Bernstein P, Peltz S W, Ross J
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Mol Cell Biol. 1989 Feb;9(2):659-70. doi: 10.1128/mcb.9.2.659-670.1989.
Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.
利用体外mRNA降解系统，我们研究了多聚腺苷酸（poly(A)）及其相关的多聚腺苷酸结合蛋白（PABP）如何影响mRNA的稳定性。在降解反应中使用的细胞提取物，通过添加过量的poly(A)竞争物或使提取物通过poly(A)-琼脂糖柱，耗尽了功能性PABP。在缺乏PABP的反应中，β-珠蛋白、氯霉素乙酰转移酶和猿猴病毒40病毒体蛋白的多聚腺苷酸化mRNA的降解速度比含有过量PABP的反应快3至10倍。向缺乏PABP的反应中添加纯化的酿酒酵母或人细胞质PABP可使多聚腺苷酸化mRNA稳定。相反，非多聚腺苷酸化mRNA的降解速率不受PABP影响，这表明poly(A)及其结合蛋白都是维持mRNA稳定性所必需的。来自大肠杆菌的非特异性单链结合蛋白不能恢复多聚腺苷酸化mRNA的稳定性，并且PABP的稳定作用被抗PABP抗体抑制。在缺乏PABP的反应中，poly(A)尾是第一个被降解的mRNA片段，这证实了poly(A)-PABP复合物保护3'区域免受核酸酶的攻击。这些结果表明，poly(A)与其结合蛋白共同发挥的一个重要功能是保护多聚腺苷酸化mRNA不被细胞核酸酶随意破坏。本文提出了一个模型来解释mRNA的稳定性如何受到其poly(A)-PABP复合物稳定性的影响。
