The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome

Abstract

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality.

Keywords: Saccharomyces cerevisiae; eukaryote-specific bridges; ribosome assembly; ribosome subunit association; stress tolerance.

Figure 1. Functional analysis of eL19 deletion mutants in vivo

A. S. cerevisiae 80S ribosome. The large subunit (60S) is shown in blue and the small subunit (40S) is grey. Proteins eL19 and eL24 forming the eB12 and eB13 bridges, respectively, are shown in red and orange. B. Domain structure of eL19. The C-terminal α-helix of eL19 (dark blue) is shown in detail. Amino acid residues forming contacts with eukaryote-specific expansion segment 6 (ES6S) of 18S rRNA and 40S subunit proteins (eS7, uS17) are coloured green and yellow, respectively. Arrows indicate the positions of the last amino acids of the respective eL19 deletion alleles. C. Close up view of the eB12 intersubunit bridge. Detailed views of contacts formed between α-helix of eL19 (dark blue) and uS17 (red), 18S rRNA ES6S (orange) and eS7 (purple) are shown. Ribosomal structures were generated by PyMol using coordinates from. D. Analysis of the functionality of eL19 deletion mutants using plasmid shuffle assay. An rpl19AΔrlp19BΔ strain (TYSC237) containing RPL19A/URA3/CEN plasmid was transformed with either an empty LEU2/CEN plasmid (indicated as vector) or plasmids harbouring wild type eL19 or indicated eL19 mutant alleles. Growth of the transformants was tested on control plates (SC-Leu) or on plates containing 5-fluoroorotic acid (SC-Leu+5FOA) which selects for yeast cells that have lost the RPL19A/URA3/CEN plasmid. Plates were incubated at 30°C for 3 days.

Figure 2. Phenotypic analysis of eL19 deletion mutants

A. Growth phenotypes of eL19 mutants. Serial dilutions of wild type (WT, TYSC144) and rpl19AΔrlp19BΔ strains carrying eL19 wild type (eL19, TYSC278) or mutant alleles (eL19_1–183, TYSC280; eL19_1–154, TYSC282; eL19_1–146, TYSC283) were spotted onto YPD medium and incubated at the indicated temperatures for 2–4 days. B. Analysis of ribosome-polysome profiles by sucrose density gradient centrifugation. The rpl19AΔrlp19BΔ strains carrying eL19 wild type or mutant alleles were grown in YPD medium at indicated temperatures. The whole cell extracts were prepared from cycloheximide treated cells and analysed in 7%–47% sucrose gradients. The absorbance at 260 nm (A260nm) was recorded. Sedimentation is from left to right. The peaks of free 60S ribosomal subunits, monosomes (80S) and polysomes are indicated.

Figure 3. Processing of pre-rRNAs is not perturbed in eL19 mutants

A. Serial dilutions of wild type (WT, TYSC144) and rpl19AΔrlp19BΔ strain carrying pGAL-RPL19 plasmid (P_GAL1::RPL19, TYSC291) were spotted onto YPGal (galactose) or YPD (glucose) medium and incubated at 30°C for 3 days. B. Analysis of ribosome-polysome profiles of cells depleted for the eL19 by sucrose density gradient centrifugation. Cells were cultured in YPGal and shifted to YPD for 12 or 24 hours. Whole cell extracts were prepared from cycloheximide treated cells and analysed in 7%–47% sucrose gradients. The absorbance at 260 nm (A260nm) was recorded. Sedimentation is from left to right. The peaks of free 40S ribosomal subunits, monosomes (80S) and polysomes are indicated. C. Structure and processing sites of the 35S pre-rRNA. The mature 18S, 5.8S and 25S rRNAs are shown as boxes. Two internal transcribed spacer sequences (ITS1 and ITS2) and two external transcribed spacer sequences (5′ ETS and 3′ ETS) are shown as lines. The cleavage sites and positions of the oligonucleotides (F and G) used for the primer extension analysis are indicated. D. Analysis of pre-rRNA processing in cells depleted for eL19 by primer extension. Total RNA was extracted from cells grown at 30°C in YPGal or shifted to YPD for up to 24 hours. Samples were subjected to primer extension analysis of 5′ ends of 27S and 25.5S pre-rRNAs. Asterisks indicate the nonspecific cleavage products of 27S pre-rRNA. The origin of the pre-rRNAs analysed by primer extension are shown. E. Analysis of pre-rRNA processing in eL19 mutants by primer extension. Total RNA was extracted from wild type (WT, TYSC144) and rpl19AΔrlp19BΔ strains carrying eL19 wild type (eL19, TYSC278) or mutant alleles (eL19_1–183, TYSC280; eL19_1–154, TYSC282; eL19_1–146, TYSC283) grown in YPD medium at 30°C or 20 °C.

Figure 4. The eB12 intersubunit bridge is required for 80S formation in vitro

Salt-washed 40S and 60S subunits were purified from rpl19AΔrlp19BΔ strains carrying eL19 wild type (eL19, TYSC278) or mutant alleles (eL19_1–183, TYSC280; eL19_1–154, TYSC282; eL19_1–146, TYSC283) grown in YPD medium at 30°C. Two A260 units of wild type 40S subunits were co-incubated with two A260 units of 60S subunits containing wild type or mutant eL19 in the presence of indicated magnesium acetate concentrations for 20 min at 30°C. Reactions at 20 mM magnesium acetate were also performed in the presence of saturating concentration of deacylated tRNA (20 mM + tRNA) to stimulate 80S formation. Ribosomal subunit association was analysed in 10%–30% sucrose gradients with appropriate magnesium acetate concentrations. Sedimentation is from left to right. The peaks of free 40S and 60S ribosomal subunits and monosomes (80S) are indicated.

Figure 5. Physiological responses of eL19 deletion mutants to different stress conditions

A. Drug resistance/sensitivity phenotypes of eL19 mutants. Serial dilutions of rpl19AΔrlp19BΔ strains carrying eL19 wild type (eL19, TYSC278) or mutant alleles (eL19_1–183, TYSC280; eL19_1–154, TYSC282; eL19_1–146, TYSC283) were spotted onto YPD medium without or with indicated antibiotics and incubated at the indicated temperatures for 3–4 days. B. Analysis of HSP12 and CTT1 transcript levels with quantitative PCR in eL19 mutants. Total RNA was extracted from rpl19AΔrlp19BΔ strains carrying eL19 wild type or mutant alleles grown in YPD medium at 30°C. The results were normalised to the geometric average of two housekeeping genes (UBC6 and ARP6) and expressed relative to the strain expressing wild type eL19. Error bars represent standard deviations of two independent measurements. C. Design of the analysis of NaCl stress survival. CFU, colony-forming unit. D. Sensitivity to salt stress of eL19 mutants. Yeast cells were grown to early exponential phase in YPD at 30°C and exposed to 0.7 M NaCl stress for 2 hours. Cell viability was measured by counting colony-forming cells on YPD plates after the salt stress relative to untreated cells. The error bars represent the standard errors of three independent experiments. Statistical significance was evaluated with the unpaired t test (*P<0.001).