The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite–multisubstrate RNA:Ψ-synthase also acting on tRNAs

Abstract

The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Ψ)-synthase acting at position 35 in U2 snRNA. However, U2 snRNA was the only potential substrate tested for this enzyme. In this work, we demonstrated that although Pus7p is responsible for the formation of only one of the six Ψ residues present in yeast UsnRNAs, it catalyzes U to Ψ conversion at position 13 in cytoplasmic tRNAs and at position 35 in pre-tRNATyr. Sites of RNA modification by Pus7p were identified by analysis of the in vivo RNA modification defects resulting from the absence of active Pus7p production and by in vitro tests using extracts from WT and genetically modified yeast cells. For demonstration of the direct implication of Pus7p in RNA modification, the activity of the WT and mutated Pus7p recombinant proteins was tested on in vitro produced tRNA and pre-tRNA transcripts. Mutation of an aspartic acid residue (D256) that is conserved in all Pus7 homologs abolishes the enzymatic activity both in vivo and in vitro. This suggests the direct involvement of D256 in catalysis. Target sites of Pus7p in RNAs share a common sequence Pu(G/C)UNΨAPu (Pu = purine, N = any nucleotide), which is expected to be important for substrate recognition. Modification of tRNAs by Pus7p explains the presence of Pus7p homologs in archaea and some bacteria species, which do not have U2 snRNA, and in vertebrates, where Ψ34 (equivalent to Ψ35 in yeast) formation in U2 snRNA is an H/ACA snoRNA guided process. Our results increase the number of known RNA modification enzymes acting on different types of cellular RNAs.