ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system
ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system
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DOI：
10.1101/gad.13.2.188
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发表时间：
1999-01-15
影响因子：
10.5
通讯作者：
Wilusz, J
中科院分区：
文献类型：
--
作者：
Ford, LP;Watson, J;Wilusz, J
我们已经开发了一个使用HeLa细胞胞质S100提取物和外源多腺苷化RNA底物的体外信使核糖核酸稳定系统，该系统再现了信使核糖核酸衰退的调节方面。冷多聚(A)竞争对手RNA的加入既激活了提取物中序列特异性的死烯基酶活性，也激活了强大的、依赖于ATP的核糖核溶解活性。底物RNA体内存在多种富含AU的元素，上调了去烯化和降解的速度。竞争分析表明，富含AU元素的RNA失稳需要反式作用因子。在体外系统中，类似于30kD的ELAV蛋白HUR特异性地结合到含有由肿瘤坏死因子-αmRNA衍生的富含AU元件的RNA上。然而，HUR与富含AU的元素的相互作用与RNA不稳定无关。有趣的是，重组ELAV蛋白特异性地稳定了富含AU元素的底物RNA周转产生的无烯基化中间产物。这些数据表明，哺乳动物的ELAV蛋白通过影响降解酶对RNA底物的访问，在调节mRNA的稳定性方面发挥作用。
We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The similar to 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.