Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U smallnuclear RNAs (snRNAs) reveals that pseudouridine synthase Pus1p exhibits adual substrate specificity for U2 snRNA and tRNA

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 on ly a few Psi residues, which are located in segments involved in intermolec ular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are u niversally modified. When yeast mutants disrupted for one of the several ps eudouridine 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 spliceosoma l UsnRNAs. Indeed, Psi(44) formation In U2 snRNA was abolished. By using pu rified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi(44) 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 bath 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.