Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping

Abstract

Ribosomal RNAs contain numerous modifications at specific nucleotides. Despite their evolutionary conservation, the functional role of individual 2'-O-ribose methylations in rRNA is not known. A distinct family of small nucleolar RNAs, box C/D snoRNAs, guides the methylating complex to specific rRNA sites. Using a high-resolution phenotyping approach, we characterized 20 box C/D snoRNA gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intraribosomal antibiotics, inhibitors of specific cellular features, as well as general stressors. Ribosome-specific antibiotics generated phenotypes indicating different and long-ranging structural effects of rRNA methylations on the ribosome. For all studied box C/D snoRNA mutants we uncovered phenotypes to extraribosomal growth inhibitors, most frequently reflected in alteration in growth lag (adaptation time). A number of strains were highly pleiotropic and displayed a great number of sensitive phenotypes, e.g., deletion mutants of snR70 and snR71, which both have clear human homologues, and deletion mutants of snR65 and snR68. Our data indicate that individual rRNA ribose methylations can play either distinct or general roles in the workings of the ribosome.

FIGURE 1.

Box C/D snoRNA deletion mutants display growth alterations in basal medium. (A) Growth curves in basal medium for wild type (filled circles), snr65Δ (crosses), and snr57Δ (open circles). (B) The three growth variables—growth lag (adaptation time), growth rate, and growth efficiency—were extracted from growth curves. The values displayed for the mutants were normalized to the wild type and expressed on a logarithmic scale, forming a logarithmic strain coefficient (LSC) (LSC = 0 means no change compared to the wild type; LSC < 0 indicates sensitivity; and LSC > 0 indicates resistance compared to the wild type). Growth lag (triangles), growth rate (circles), and growth efficiency (squares) are shown; filled symbols indicate values that are significantly different (P < 0.001) from the wild type.

FIGURE 2.

Ribosomal inhibitors differently affect growth of the snoRNA deletion mutants. The ribosomal inhibitors neomycin, paromomycin, anisomycin, cycloheximide, and puromycin were administered at concentrations that elicited a <20% growth rate reduction in the wild type. The growth of the snoRNA mutants in the presence of the ribosomal inhibitor was normalized for any general growth defects for the mutants in basal medium, forming the logarithmic phenotypic index (LPI): LPI = LSC_inhibitor − LSC_basal. (A) Two-way hierarchical clustering of LPI for the three growth variables and inhibitors. Only statistically significant (P < 0.001) values are displayed in color; LPI < 0 (green) and LPI > 0 (red). Two of the clusters have been indicated; the snoRNA deletions that confer mainly improved growth compared to the wild type are indicated as resistant (red), and the ones that confer mainly growth worse than the wild type are indicated as sensitive (green). (B) 3D structural model of the rRNA portion of the ribosome with the small subunit to the left (SSU) and the large subunit to the right (LSU). Positions of the 2′-O-ribose methylations are indicated; red and green indicate clustering in A. The two functionally important domains, peptidyl transfer center and decoding center, are indicated with gray circles, and a tRNA molecule that stretches between these two centers is indicated in blue.

FIGURE 3.

The snoRNA deletion mutants respond differently to wide array of extraribosomal growth inhibitors and stresses. A one-way hierarchical cluster on LPI is displayed; only statistically significant (P < 0.001) better (red) and worse (green) growth than the wild type is indicated. Clustering was performed on all data, but the three different growth variables are displayed separately. The r values by the tree to the left indicate the Pearson correlation at that branch point. The corresponding human rRNA methylations are indicated after the strain name.

FIGURE 4.

Certain mutants display a great number of sensitive phenotypes. The number of significantly (P < 0.001) sensitive phenotypes is indicated for each of the three growth variables separately: growth lag (black), growth rate (white), and growth efficiency (gray).