Sabatini R, Hajduk S L
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham 35294, USA.
J Biol Chem. 1995 Mar 31;270(13):7233-40. doi: 10.1074/jbc.270.13.7233.
RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is thought to be directed by guide RNAs and may proceed via a chimeric guide RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprotein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylylated proteins of 50 and 57 kDa co-sediment on glycerol gradients with RNA ligase activity as components of the ribonucleoprotein particles. The two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addition of ligatable RNA substrate with the concomitant release of AMP and by the addition of pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate results in an accumulation of the adenylylated RNA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable analogs indicates that both RNA ligase and chimera-forming activities require alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera formation. These results strongly suggest the involvement of RNA ligase in in vitro chimera formation and support the cleavage-ligation mechanism for kinetoplastid RNA editing.
布氏锥虫中的RNA编辑导致几种线粒体mRNA中尿苷残基的添加和缺失。编辑被认为是由引导RNA指导的，并且可能通过嵌合引导RNA/mRNA中间体进行。我们之前已经表明，形成嵌合体的活性与19S和35 - 40S线粒体核糖核蛋白颗粒（RNP）一起沉降。在本报告中，我们研究了RNA连接酶在体外嵌合分子产生中的作用。两种50 kDa和57 kDa的腺苷酸化蛋白作为核糖核蛋白颗粒的组分，在甘油梯度上与RNA连接酶活性一起沉降。这两种腺苷酸化蛋白在序列上不同，并且含有通过磷酰胺键连接的AMP。通过添加可连接的RNA底物伴随着AMP的释放以及通过添加焦磷酸产生ATP，这两种蛋白都会去腺苷酸化。用不可连接的RNA底物孵育会导致腺苷酸化RNA中间体的积累。这些实验将腺苷酸化蛋白鉴定为RNA连接酶。线粒体RNA连接酶释放AMP也与嵌合体形成同时发生。不可水解类似物的抑制作用表明，RNA连接酶和形成嵌合体的活性都需要ATP的α-β键水解。连接酶的去腺苷酸化抑制嵌合体形成。这些结果强烈表明RNA连接酶参与体外嵌合体形成，并支持动质体RNA编辑的切割-连接机制。
