Behm-Ansmant Isabelle, Massenet Séverine, Immel Françoise, Patton Jeffrey R, Motorin Yuri, Branlant Christiane
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Nancy I, Faculté des Sciences, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.
RNA. 2006 Aug;12(8):1583-93. doi: 10.1261/rna.100806. Epub 2006 Jun 27.
Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.
小鼠假尿苷合酶1（mPus1p）是首个被克隆并进行生化特性鉴定的脊椎动物RNA：假尿苷合酶。此前发现mPus1p可在体外产生的酵母和人tRNA中的第27、28、34和36位催化假尿苷（Ψ）的形成。另一方面，已表明同源的酿酒酵母scPus1p蛋白可修饰tRNA中的七个尿苷残基（26、27、28、34、36、65和67）以及U2 snRNA中的U44。在本研究中，我们在缺乏scPus1p的酵母细胞中表达mPus1p，并研究U2 snRNA和几种酵母tRNA的修饰情况。我们的数据表明，在这些体内条件下，小鼠酶可有效修饰酵母U2 snRNA的第44位以及tRNA的第27、28、34和36位。然而，未观察到mPus1p的tRNA：Ψ26合酶活性。此外，我们发现scPus1p和mPus1p在体内和体外对细胞质tRNAArg(ACG)的第1位均具有一种此前未被鉴定的活性。这种修饰可发生在成熟tRNA以及具有5'和/或3'延伸的前体tRNA中。因此，我们鉴定出了具有酵母tRNA：Ψ合酶最后缺失活性之一的蛋白。此外，我们的结果揭示了mPus1p在tRNA第30位具有一种scPus1p所不具备的额外活性。
