Cruz-Reyes J, Sollner-Webb B
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8901-6. doi: 10.1073/pnas.93.17.8901.
We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.
我们利用四对mRNA-引导RNA（gRNA），这些gRNA可指定在编辑位点1处删除2、3或4个尿嘧啶（U）残基，并结合线粒体提取物，研究了布氏锥虫ATP酶6 mRNA在体外进行精确RNA编辑的机制。该提取物不仅能催化删除特定数量的U残基，还表现出一种新型的内切核酸酶活性，该活性以gRNA定向的方式切割输入的前体mRNA，精确地在RNA编辑的简单酶促模型中预测的磷酸二酯键处切割。这个切割位点与基于嵌合体的编辑机制不一致。随后，上游切割产物3'端存在的待删除U残基显然是通过3' U特异性核酸外切酶去除的，而不是通过末端U转移酶的反向反应去除。然后，RNA连接酶可以通过新形成的5'磷酸（P）和3'羟基（OH）末端连接mRNA的两半，从而产生在第一个编辑位点处经过忠实编辑的mRNA。这个产生的部分编辑的mRNA随后可以在编辑位点2进行精确的、gRNA定向的切割，同样正如酶促编辑模型所预测的那样。所有这些酶活性都与U删除活性一起共分级分离，并且可能存在于一个单一的复合物中。这些数据表明，每一轮编辑都是一个四步过程，包括（i）gRNA定向在与gRNA碱基配对区域紧邻的5'处的键切割前体mRNA，（ii）在上游mRNA一半的3' OH处删除U或添加U，（iii）连接mRNA的两半，以及（iv）在正确编辑的位点和指导下一个编辑位点后续核酸酶切割的gRNA之间形成额外的碱基配对。
