Byrne E M, Connell G J, Simpson L
Howard Hughes Medical Institute, UCLA School of Medicine, 90095-1662, USA.
EMBO J. 1996 Dec 2;15(23):6758-65.
Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.
引导RNA（gRNA）被认为可介导动质体原生动物中线粒体mRNA的尿苷（U）添加/缺失编辑。据推测，这些U要么通过两次连续的切割-连接或酯交换反应直接来源于三磷酸尿苷（UTP），要么通过相同机制来源于gRNA的3'末端。我们已经证明，在来自热带利什曼原虫的线粒体提取物中孵育的预编辑mRNA的特定编辑位点上，gRNA依赖的U插入反应。U插入的主要数量由添加的gRNA中引导核苷酸的数量决定，并且gRNA-mRNA锚定双链体的形成对于活性是必需的。需要UTP和ATP的α-β键水解，并且在50-100 mM KCl以上活性受到抑制。在没有添加同源gRNA的情况下，会发生多达约13个U的不依赖gRNA的插入；这种活性的程度受编辑区域上游和下游序列的影响。肝素抑制不依赖gRNA的U插入活性，而对依赖gRNA的活性没有影响。封闭gRNA的3'羟基对依赖gRNA的U插入活性影响很小。这些数据与一个切割-连接模型一致，其中U直接来源于UTP。
