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 yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial 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 residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 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 YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability.
Ansmant
I
I
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.
Massenet
S
S
Grosjean
H
H
Motorin
Y
Y
Branlant
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002455:Cell Division; D005656:Fungal Proteins / Q000378:metabolism; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D011560:Pseudouridine / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D000077278:RNA, Mitochondrial; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D014529:Uridine / Q000378:metabolism
D005656:Fungal Proteins; D000077278:RNA, Mitochondrial; D012335:RNA, Ribosomal; D012342:RNA, Small Nuclear; D011560:Pseudouridine; D012313:RNA; D012343:RNA, Transfer; D019751:Intramolecular Transferases; D014529:Uridine