Spinelli S L, Consaul S A, Phizicky E M
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, New York 14642, USA.
RNA. 1997 Dec;3(12):1388-400.
tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.
tRNA剪接在酵母、人类以及大概所有真核生物中都是必不可少的。酵母tRNA剪接的前两个步骤，即通过核酸内切酶切除内含子以及通过tRNA连接酶连接外显子，会留下一个带有2'-磷酸的剪接接头。生化分析表明，酵母中这种磷酸的去除是由一种高度特异性的2'-磷酸转移酶催化的，该酶将磷酸转移到NAD上以形成ADP-核糖1''-2''环磷酸。2'-磷酸转移酶的催化活性由酿酒酵母中的一个单一必需基因TPT1编码。我们在此表明，Tpt1蛋白在体内负责tRNA剪接的去磷酸化步骤，因为在非允许生长期间，条件致死的tpt1突变体积累了来自已知会被剪接的八种不同tRNA种类的2'-磷酸化tRNA。我们还表明，这些tRNA中的几种在剪接接头残基处修饰不足，该残基总是位于反密码子下游一个碱基的超修饰位置。这一结果与之前的结果一致，即超修饰位置的修饰发生在tRNA加工途径中的内含子切除之后，并且意味着修饰通常在体内tRNA剪接的去磷酸化步骤之后进行。
