Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

Abstract

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

Figure 1. Model of the hypothetical location of eIF3 on the S. cerevisiae small ribosomal subunit.

(Upper panel) The Cryo-EM reconstruction of the 40S subunit is shown from the solvent side with ribosomal RNA represented as tubes. Ribosomal proteins, with known homologs and placement, are shown as pink cartoons and labeled (adapted from [40]). The positions of helices 16–18 of 18S rRNA, and ribosomal proteins RACK1/ASC1, RPS2, 3, and 20 are highlighted in bold. The position of RPS0, the subject of this study, is highlighted in bold and underlined. The mRNA entry channel is designated by an asterisk. (Lower panel) Hypothetical location of S. cerevisiae eIF3 on the back side of the 40S subunit based on the data presented in this study and elsewhere, including the interactions between RPS0 and the NTD of a/TIF32 (in bold and underlined); the c/NIP1-CTD and RACK1/ASC1; RPS2 and j/HCR1; helices 16–18 of 18S rRNA and RPS2 and 3 with the a/TIF32-CTD; and RPS3 and 20 and g/TIF35 (all in bold). The 3D structural model of the c/NIP1-CTD/PCI domain and the X-ray structure of the yeast i/TIF34 – b/PRT1-CTD complex were used to replace the original schematic representations of the same molecules. The yellow lines represent mRNA.

Figure 2. Small ribosomal protein RPS0A interacts with the region spanning amino acid residues 200 and 400 of eIF3a/TIF32 via its extreme C-terminal tail in vitro.

(A) Full-length RPS0A fused to GST (lane 3) and GST alone (lane 2) were tested for binding to ³⁵S-labeled wt a/TIF32-NTD [amino acid residues 1–400] and its N- and C-terminal halves; 10% of input amounts added to each reaction is shown in lane 1 (In). The schematic to the right illustrates two discernible regions of the a/TIF32-NTD, one of which promotes reinitiation after translation of short uORFs by contacting specific mRNA regions preceding these uORFs , , and the other interacts with RPS0A. (B) GST fusions of two consecutive segments of the a/TIF32-NTD in NTD-Δ8 [residues 200–400] and NTD-N2–200 [201–400] in lanes 3 and 4, respectively, or GST alone (lane 2) were tested for binding to the purified wt 40S ribosomal subunits. Lane 1 (In) contains 2.5% of input amounts of 40S subunits added to each reaction mixture. Binding to 40S ribosomes was detected by Western blotting with antibodies against ASC1 and RPS22. (C) Full-length RPS0A (lane 3) and its C-terminal truncations (lanes 4–6) fused to GST, and GST alone (lane 2), were tested for binding to ³⁵S-labeled wt a/TIF32; 10% of input amounts added to each reaction is shown in lane 1 (In); short and long exposures are displayed as indicated.

Figure 3. Conditional depletion of RPS0A significantly decreases translation initiation rates.

(A) The RPS0A-depletion ceases the growth of mutant cells in non-permissive conditions. Strain YID16 (rps0aΔ rps0bΔ YEpMET-RPS0A-U) bearing the RPPS0A WT allele under control of MET3 promoter was spotted in five serial 10-fold dilutions on SD medium +/− methionine and incubated at 30°C for 2.5 days. Growth curves of the same cells grown in liquid SC media lacking methionine at 30°C to an optical density (OD₆₀₀) of 0.15, split into two halves, and further cultivated under the permissive (Met−) and non-permissive (Met+; with 20 mM methionine) conditions for the indicated time intervals at which OD₆₀₀ readings were taken. (B) Rapid depletion of the RPS0A protein in the non-permissive media. The YID16 cells were grown in liquid SC media lacking methionine at 30°C to OD₆₀₀ of 0.3, split into two halves, grown without (lane 1) or with (lanes 2–4) methionine for the indicated time intervals, and WCEs were prepared and subjected to Western analysis using antibodies against the indicated proteins. (C) Rapid depletion of RPS0A dramatically reduces the polysome content. The YID16 cells were cultured under the same conditions as in panel B (the 8 hr interval was chosen for Met⁺ culture) and treated with cycloheximide (5 mg/100 ml) for 5 min prior to harvesting. WCE were prepared and subsequently separated on a 5%–45% sucrose gradient by centrifugation at 39,000 rpm for 2.5 h. The gradients were collected and scanned at 254 nm to visualize the ribosomal species. Positions of 40S, 60S and 80S species are indicated by arrows and polysome to monosome (P/M) ratios are given above the profiles.

Figure 4. RPS0A mediates in vivo association of eIF3 and its associated eIFs with the 40S ribosomal subunit.

The same as in Figure 3C, except that the cells were treated with 2% formaldehyde instead of cycloheximide and WCEs were separated on a 7.5%–30% sucrose gradient by centrifugation at 41,000 rpm for 5 h. Proteins from the collected fractions were subjected to Western analysis using antibodies against the proteins listed on the right-hand side of the blots. An aliquot of each WCE was analyzed in parallel (In, input); fractions 1–5, 6–9, and 10–12 were combined. Rectangles indicate fractions where the 43S and 48S pre-initiation complexes sediment (40S); percentages indicate the relative amount of the 40S species in the cells grown under non-permissive versus permissive conditions. Amounts of the each individual factor in the pooled fractions from three independent experiments were quantified by fluorescence imaging, combined and the percentage representation of the signal corresponding to the Top (1–5), Middle (6–9) or 40S (10–12) fractions was calculated and plotted.

Figure 5. The CTT of RPS0A, making a direct contact with the a/TIF32-NTD, contributes to the RPS0A role in anchoring eIF3 to the small ribosomal subunit.

(A) The same as in Figure 4, except that the YID16 cells transformed with a high copy plasmid bearing either wt RPS0A-FLAG (in TK156) or rps0A-ΔCTT-FLAG (in TK157) alleles under control of the RPS28 promoter, as the only source of the RPS0A protein product, were grown at 37°C and subsequently subjected to the 2% formaldehyde cross-linking procedure; dt indicates doubling times measured at 37°C. (Expression levels of both FLAG-tagged RPS0A protein variants in TK156 and 157 strains are shown in the right-handed panel). (B) Small ribosomal subunits containing C-terminally truncated RPS0A do not compete well for eIFs recruitment with native ribosomes. A FLAG-tag affinity purification of 40S ribosomes and its associated eIFs from WCEs prepared from the H2880 wt strain overexpressing either wt or C-terminally truncated RPS0A-FLAG followed by Western blotting. (Expression levels of both FLAG-tagged proteins in high copy number in H2880 are shown in the right-handed panel).