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

Abstract

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.

Figure 1

Sequence alignment of E.coli proteins from the RluA family (RluA, RluC and RluD) and S.cerevisiae YLR165c-encoded protein. The scale in amino acid residues is presented at the top. The three motifs (I, II and III) conserved in the RluA and RsuA families of RNA:Ψ-synthases are shown at the bottom. Identical and conserved residues are shaded. Aspartic acid residue mutated to alanine in the conserved motif II is indicated by an asterisk. Amino acid sequences of Motif II in different RNA:Ψ-synthase families are indicated at the bottom.

Figure 2

UsnRNA pseudouridylation pattern in the ΔYLR165c S.cerevisiae strain. Primer extension analysis was performed for U1 (A), U2 (B) and U5 (C) snRNAs from a total RNA fraction of the wild-type (WT) and ΔYLR165c S.cerevisiae strains. CMCT modifications were done for 1, 10 and 20 min (lanes 2, 3 and 4 respectively), experimental conditions are described in Materials and Methods. In lanes 3 and 4, the CMCT-modified RNA was subjected to an alkaline treatment at pH 10.4. A control extension experiment was made without addition of CMCT (lane 1). Lanes U, G, C and A correspond to the RNA sequencing ladder. Nucleotide positions, starting from the 5′-terminal nucleotide, are indicated on the right. The reverse transcription stops, corresponding to Ψ residues in U1, U2 and U5 snRNAs, are indicated by arrows on the left.

Figure 3

Ψ at position 2819 in yeast mitochondrial 21S rRNA disappears upon deletion of the YLR165c ORF. Primer extension analysis of the CMCT-modified S.cerevisiae mitochondrial 21S rRNA in a total RNA fraction from wild-type (WT) or ΔYLR165c S.cerevisiae strains, for 1 (lanes 2 and 8), 10 (lanes 3, 5, 9, 11) and 20 min (lanes 4, 6, 10, 12). A control extension experiment was made without addition of CMCT (lanes 1 and 7). The experimental conditions are the same as in Figure 2. The reverse transcription stop, corresponding to residue Ψ₂₈₁₉, is indicated by an arrow on the left. Absence of this stop in the reverse transcription pattern of the 21S rRNA from the ΔYLR165c strain (lanes 5 and 6) indicates the absence of residue Ψ₂₈₁₉. Primer extension analysis of the CMCT-modified S.cerevisiae mitochondrial 21S rRNA in a total RNA fraction from the ΔYLR165c strain transformed with plasmid p413TEF-YLR165c (lanes 9 and 10) and with a plasmid bearing mutated YLR165c ORF (lanes 11 and 12).

Figure 4

The three modified nucleotides of the S.cerevisiae mitochondrial 21S rRNA and their counterpart in E.coli 23S rRNA. Only the part of Domain V containing the three modified residues is represented according to the 2D structure drawn by Sirum-Connolly et al. (44). The positions of the three modified nucleotides are shown, the numbers of their counterparts in E.coli are indicated in brackets (43–45). Nucleotides are numbered starting from 5′-terminal. Helices are numbered according to Leffers et al. (50).