Identification of the Saccharomyces cerevisiae RNA : pseudouridine synthase responsible for formation of Psi(2819) in 21S mitochondrial ribosomal RNA

So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeas t Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochond rial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi -synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified, Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs ) encoding putative RNA:Psi-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi r esidue normally present in the fully matured mitochondrial rRNAs (Psi(2819) in 21S rRNA) was missing, while Psi residues at all the tested pseudouridy lation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitocho ndrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165 c-encoded protein, indicating the direct role of the YLR165c protein in Psi (2819) synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogen eity the YLR165c ORF was renamed PUSS and the corresponding RNA:Psi-synthas e Pus5p. As already noticed for other mitochondrial RNA modification enzyme s, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819) in mitochondrial 21S rRNA is not essentia l for cell viability.