A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast

Abstract

Ribosomal RNAs contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two types: 2'-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications. The 2'-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively. The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2'-O-ribose methylations or pseudouridylations. Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Psi at position 2919 within the LSU rRNA. Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA. Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell.

FIGURE 1.

Characterization of snR191. (A) Northern blot analysis of snR191. Total RNAs (10 μg) from wild-type strain (lane 1) or Δdbr1 strain (lane 2), separated on a 5% acrylamide-urea gel, were hybridized, after transfer to a nylon membrane, with [³²P] 5′ end-labeled oligonucleotide CS9 (see Materials and Methods). The marker is pBR322 DNA digested with MspI and [³²P] 5′ end-labeled. (B) The 5′ end of snR191 was mapped by reverse transcription using [³²P] 5′ end-labeled oligonucleotide GB213 as primer and total RNAs extracted from Mat a or Mat α (as indicated) wild-type BMA64 strains as template. (Lanes C,T,A,G) Dideoxy sequencing reactions using the same oligonucleotide primer as for reverse transcription and, as template, DNA amplified by PCR using primers MFR207 and MFR208 (see Materials and Methods). The reverse transcription and sequencing reactions were performed as previously described (Saveanu et al. 2001). An arrow indicates the 5′ start position. (C) Schematic representation of the NOG2 gene indicating the relative positions of the different oligonucleotides used for RNA analyses (arrows). The GB242 oligonucleotide overlaps the snoRNA deletion represented by the dotted line within the corresponding arrow. (White boxes) exons; (gray and black boxes) intron sequences. The black box represents the small RNA sequence.

FIGURE 2.

Structure of snR191. (A) Alignment of the Saccharomyces cerevisiae snR191 sequence with that of other yeast species and human hU19. (S. ce) S. cerevisiae; (S. ba) Saccharomyces bayanus (EMBL accession number AL398402); (K. ma), Kluyveromyces marxianus (EMBL accession number AL422984); (hU19) human hU19 (Kiss et al. 1996). Note that the S. bayanus and K. marxianus sequences are issued from the Génolevure project (Souciet et al. 2000) and thus correspond to single reads that are prone to contain some errors or ambiguities (e.g., M stands for A or C). The alignments were performed with the program clustalW (Thompson et al. 1994). Nucleotides conserved in all four sequences are shaded in black, nucleotides conserved in three species are shaded in gray. H and ACA boxes are indicated. Guiding sequences for pseudouridylations are labeled AA′ and BB′. (B, C, and D) Secondary structure of yeast snR191s. Among all snR191 potential secondary structures predicted using the MFOLD program (Walter et al. 1994), the ones that are conserved between S. cerevisiae (B), S. bayanus (C), and K. marxianus (D) are shown. (E) Secondary structure of human hU19 RNA as determined in Kiss et al. (1996). The potential guide sequences are boxed and labeled AA′ and BB′ in the S. cerevisiae structure in B. In C and D, and E, boxes represent conserved nucleotides with regard to S. cerevisiae. The H and ACA motifs are indicated. Note that the 3′ end of snR191 has been experimentally determined with a precision of ±2 nt from the determined 5′ end and the measured length of the RNA (274 ± 2 nt). The actual position of the 3′ end of snR191 is thus known at ±2 nt. Nevertheless, using a length of 274 nt, as presented on the figure, leads to predict a 3′ end 3 nt downstream of the ACA box, precisely as in the consensus H/ACA snoRNA structure (Ganot et al. 1997b).

FIGURE 3.

Mapping pseudouridines targeted by snR191. Primer extension with [³²P] 5′ end-labeled oligonucleotide GB150 (complementary to 25S rRNA; see Materials and Methods) and RNAs from wild-type (LMA5–1D) and Δintron (LMA194) strains treated (+) or not treated (−) with CMC (see Materials and Methods). (Lanes C,T,A,G) Dideoxy sequencing reaction using the same oligonucleotide and, as template, 25S rDNA amplified by PCR using primers GB130 and GB131. The positions of pseudouridines are marked by Ψs and the corresponding snoRNA guides, when known, are indicated between brackets.

FIGURE 4.

Positions of the two highly conserved Ψs in the large subunit rRNA structure. The position of the two highly conserved Ψs is represented by red spheres (and pointed to by arrows) on the crystal structure (5.5 Å resolution) of the large ribosomal subunit of Thermus thermophilus as determined in Yusupov et al. (2001) (PDB identification number: 1GIY). A backbone representation is used, with rRNAs in blue and proteins in orange. The rRNA regions that are in contact with tRNAs, according to Yusupov et al. (2001), are shown in violet. The structure was displayed using the program RasMol v2.6 (Sayle and Milner-White 1995). (A) View from the small subunit binding face. The approximate regions of the A, P, and E tRNA sites, as well as of helix 69 that carries the two highly conserved Ψs, are pointed to by straight lines. (B) View from the left side of the large subunit.

FIGURE 5.

Competition assay between wild-type cells and cells lacking snR191. The amount of wild-type cells (LMA5–1D strain) relative to cells lacking snR191 (LMA247 strain) during the course of cultures is measured by the relative quantification of PCR products arising from LMA5–1D (wild type) cells (PCR product generated by the GB241 and GB243 oligonucleotides) or LMA247 (ΔsnR191 mutant) cells (PCR product generated by the GB241 and GB242 oligonucleotides) (see Materials and Methods). (A) PCR samples from standard (top) or competition experiments (bottom) were loaded on an 8% acrylamide-urea gel. The figure shows PhosporImager images of the gels. For calibration, standard reference values were generated by quantifying, with a PhosphorImager, the relative amount of the PCR products generated from known mixed fractions of wild-type and ΔsnR191 yeast cells. The evolution of the fraction of wild-type cells during the competition experiment was measured by quantification of the PCR products and normalization using the values determined in the standard experiment. (B) The averaged quantifications of wild-type over mutant cells (% of wild-type cells), averaged from six independent cultures, are plotted to the number of generations. Standard deviations are shown.