Murthy K G, Park P, Manley J L
Department of Biological Sciences, Columbia University, New York, NY 10027.
Nucleic Acids Res. 1991 May 25;19(10):2685-92. doi: 10.1093/nar/19.10.2685.
We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.
我们开发了一种针对存在于HeLa细胞核提取物中的外切核糖核酸酶的检测方法，该酶可降解带帽但不带帽的RNA底物，并利用它对这种活性进行了部分纯化和表征。将不同大小（37 - 317 nt）的带帽和不带帽转录本与分级分离的核提取物一起孵育，在所有情况下，带帽RNA都是稳定的，而它们不带帽的对应物则被完全降解。当反应混合物中包含帽类似物时，未检测到活性变化，这表明带帽RNA的稳定性不是由于帽结合蛋白。已证明该外切核糖核酸酶对RNA具有特异性，并且对含有5'-羟基或5'-磷酸化末端的底物都能持续发挥作用。产物主要是5'-单核苷酸，在任何反应时间点都未观察到可检测到的中间体。沉降分析表明，该核酸酶的天然大小为7.4S或约150 kDa。有趣的是，发现核苷三磷酸对于不带帽RNA的特异性和完全降解是必需的。最后，对部分纯化的酶进行微球菌核酸酶（MN）预处理会抑制其活性。由于几个对照表明这不是由于MN的非特异性作用，这一发现表明该外切核糖核酸酶含有一种必需的RNA成分。
