Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae

Abstract

The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62-K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62-K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62-K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62-K70 segment of Rps26 and the 5' untranslated region of mRNA. The data suggested a specific role of the Y62-K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62-K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5' untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62-K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.

Keywords: 40S subunit; Saccharomyces cerevisiae; eukaryote-specific motif; mutagenesis; ribosomal protein; ribosome assembly; translation initiation; yeast genetics.

FIG 1

Structural features of Rps26a. (A) Crystal structure of S. cerevisiae Rps26 (adapted from PDB 3U5G and drawn using PyMOL Molecular Graphics System [Schrodinger, Germany]). The β-strand containing the eukaryote-specific motif 62-YALPKTYNK-70 (22) and its mirror sequence on the opposite β-strand are shown in green and blue, respectively. The COOH and NH₂ termini are indicated. (B) Multiple alignment of Rps26 proteins from S. cerevisiae, Homo sapiens, and Staphylothermus marinus. The eukaryote-specific motif and its mirror sequence are shown in green and blue, respectively. Sequence regions of low similarity, located predominantly within both β-strands and in the α-helical region in the middle of the molecule, are indicated in boldface. The amino acid numbers are based on the sequence of S. cerevisiae Rps26a.

FIG 2

Growth analysis of S. cerevisiae strains expressing wild-type Rps26a or Rps26^5A. Yeast strains were cultivated in YPD at 30°C, 40°C, or 15°C or in YPD plus 100-µg/ml paromomycin (PM), 10 mM DTT, 1 M NaCl, or 50 mM Tris-HCl (pH=8) at 30°C. Plates were incubated for 2 to 5 days depending on growth conditions and supplements.

FIG 3

Viability of yeast strains expressing different variants of Rps26. (A) Strains of S. cerevisiae Δrps26a Δrps26b carrying pRPS26a (URA3) and either pRS313 (vector), pRS313-Rps26a, or pRS313-Rps26a^del9 (see Table S1) were grown on single 5-FOA plates for 3 to 4 days at 30°C. (B) Total yeast extracts were prepared from wild-type yeast (WT), wild-type yeast expressing c-myc-tagged Rps26a (WT+Rps26a::c-myc) or c-myc-tagged Rps26a^del9 (WT+Rps26a^del9::c-myc), or Δrps26a Δrps26b yeast expressing c-myc-tagged Rps26a (Rps26::c-myc). Aliquots were analyzed by Western blotting using anti-c-myc antibody and anti-Sse1 antibody as a loading control.

FIG 4

Polysome profile analysis of yeast cells producing Rps26a or Rps26a^5A at 30°C or 15°C. S. cerevisiae variants and growth temperatures are indicated. Ribosome sedimentation was controlled by monitoring A₂₅₄. Peaks showing 40S, 60S, 80S, and polysome (Poly) contents are indicated.

FIG 5

Viability of yeast strains expressing human Rps26-Hs. (A) Serial dilutions of S. cerevisiae Δrps26a Δrps26b complemented by plasmid-encoded yeast Rps26a or human Rps26-Hs were spotted onto YPD plates and cultivated for 3 days at 30°C. (B) Yeast Δrps26a Δrps26b complemented with Rps26a or human Rps26-Hs was grown at 30°C to mid-log phase in SDex liquid medium and then analyzed as described in Materials and Methods. Ribosome sedimentation was monitored at 254 nm. Peaks showing 40S, 60S, and 80S subunits and polysome (Poly) contents are indicated.

FIG 6

Ratios of 40S/60S subunits in Δrps26a Δrps26b strains complemented by wild-type Rps26a, Rps26a^5A, or Rps26-Hs. (A) Total extracts were generated as described in Materials and Methods and analyzed by Western blotting with anti-Rps9 (40S ribosomal subunit marker) and anti-Rpl24 (60S ribosomal subunit marker) antisera. The results from one representative experiment are shown. (B) Agarose gel analysis of total rRNA isolated from strains as indicated in panel A. (C) Detected bands shown in panel A were quantified using ImageJ (57). The relative ratios of Rps9/Rpl24 are the means of the results of 2 independent experiments with 3 replicates each. The standard deviations are indicated. The band intensities of Rps9 and Rpl24 were determined in the same lane. The differences between the subunit ratios in Rps26a, Rps26a^5A, and Rps26-Hs strains are not significant. (D) rRNA bands shown in panel B were quantified using ImageJ (57). The relative ratios of 18S/28S rRNA are the mean results from 2 independent RNA preparations with 4 replicates each. The standard deviations are indicated. The difference between the ratios of 18S/28S rRNA in Rps26a and Rps26a-Hs is significant (P < 0.05, n = 8). (E) 40S and 60S peaks in polysome profiles of strains grown at 30°C (shown in Fig. 4 and 5) were subjected to quantification via ImageJ software (57). Shown are the relative ratios of the 40S/60S subunits in profiles from Rps26a, Rps26^5A, and Rps26a-Hs strains. The data represent the mean results from 3 independent experiments. The standard deviations are indicated. Example of profiles are shown in Fig. S3 in the supplemental material.