Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy

Abstract

Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2'-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains-the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.

Figure 1.

Modification map of S. cerevisiae rRNA. (A) Secondary structure of the small subunit (SSU) and large subunit (LSU) rRNAs with Nm and Ψ modifications. The modification-rich regions featured in this study are boxed. (B) Modifications in the decoding region in the SSU. The partial sequences of five stem–loop regions implicated in decoding are shown. Modifications are identified with yeast rRNA position numbers, and names of the corresponding guide snoRNAs are given in parentheses. A gray triangle indicates a modification in helix 31 that is not implicated in decoding. (C) Modifications in the ASF region in the LSU. Two flanking helices examined are also shown. Gray dots and triangles indicate modifications not examined in this study. (D) Modifications in H69 in the LSU. Flanking stem–loop structures are also given.

Figure 2.

Sensitivity of rRNA modification mutants to translational inhibitors. WT and mutant strains lacking individual modification were spotted as 10-fold dilutions on YPD and YPD containing the indicated antibiotic media. Cells were incubated for 3 days at 30°C. (A) Loss of m¹acp³Ψ1191 modification in the DC leads to doxycycline resistance. (B) Loss of modification in H69 induces aminoglycoside resistance.

Figure 3.

Two modifications in the DC are in close proximity with tRNA and mRNA at the P site. 3D representation of the rRNA bases of interest mapped onto E. coli P-region. Modified nucleotides and the corresponding guide snoRNAs are shown in various colors, mRNA is shown in blue and P-tRNA is shown in orange.

Figure 4.

Modifications within H69. 3D representation of the rRNA nucleotides of interest mapped onto E. coli H69. Modified nucleotides and the corresponding guide snoRNAs are shown in color, mRNA is shown in blue and A-site tRNA is shown in yellow.