Massenet S, Motorin Y, Lafontaine D L, Hurt E C, Grosjean H, Branlant C
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR7567 CNRS-UHP, Faculté des Sciences, 54506 Vandoeuvre-les-Nancy Cédex, France.
Mol Cell Biol. 1999 Mar;19(3):2142-54. doi: 10.1128/MCB.19.3.2142.
Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.
通过化学图谱法确定了酿酒酵母剪接体U小核RNA（UsnRNAs）中的假尿苷（Ψ）残基的位置。与脊椎动物的UsnRNAs不同，酿酒酵母的UsnRNAs只含有少数Ψ残基，它们位于参与分子间RNA-RNA或RNA-蛋白质相互作用的片段中。在这些位置，UsnRNAs普遍发生修饰。当检测破坏了几个假尿苷合酶基因（PUS1、PUS2、PUS3和PUS4）之一或rRNA-假尿苷合酶Cbf5p缺失的酵母突变体的UsnRNA Ψ含量时，发现只有Pus1p活性的丧失会影响剪接体UsnRNAs中Ψ的形成。事实上，U2 snRNA中Ψ44的形成被消除了。通过使用纯化的Pus1p酶和体外产生的U2 snRNA，本文证明Pus1p能催化酿酒酵母U2 snRNA中Ψ44的形成。因此，Pus1p是迄今为止鉴定出的首个具有双重底物特异性的UsnRNA假尿苷合酶，它既能作用于tRNA，也能作用于U2 snRNA。由于rRNA-假尿苷合酶Cbf5p的缺失对UsnRNA Ψ含量没有影响，酿酒酵母UsnRNAs中Ψ残基的形成不依赖于Cbf5p-snoRNA引导机制。
