Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome

Figures

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  Figure 1.. Effects of S278E RACK1 on ribosome rotation and translational output (A) Western blot analysis of free and ribosomal fractions. L, lysate; L.E., long exposure. Representative of 3 independent biological replicates. (B) Ab initio 3D classification of 80S ribosomes from WT RACK1 and S278E RACK1 purifications reveals a shift toward 40S-rotated, eEF2-bound particles in the presence of S278E RACK1. (C) Reconstruction of rotated ribosomes from S278E RACK1 purifications reveal densities ascribed to eEF2, E-site tRNA, EBP1, and a nascent chain. (D) Quantification of RACK1 protein levels (n = 22) and 35S-methionine/cysteine (35S-Met/Cys) incorporation (n ≥ 4). Bars represent SEM; ****p ≤ 0.0001; two-way ANOVA with Sidak’s multiple comparison test. (E) Densitometry-based quantification of the indicated protein levels (n = 4). Bars represent ± SEM; ***p = 0.0004; N.S., not significant; two-way ANOVA with Sidak’s multiple comparison test. See also Figures S1–S5.

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  Figure 2.. Negative charge in the RACK1 loop confers resistance to ribosome-targeting drugs (A) Schematic of the ribosome and target sites of inhibitors used in (B)–(D). (B–D) 35S-Met/Cys-labeling gels (top panel) and western blot analysis (bottom panels) of cells treated with the indicated concentrations of anisomycin (ANS; B), cycloheximide (CHX; C), or emetine (EME; D). Red bars/arrows highlight examples of proteins whose synthesis is repressed by inhibitors. Green arrows highlight examples of proteins whose synthesis is sustained. P-p38, phosphorylated p38; P-JNK, phosphorylated JNK; L.E., long exposure. Representative of 3 independent biological replicates. See also Figure S5.

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  Figure 3.. A negatively charged RACK1 loop affects the ribosomal E-site and RQC reporter activity (A and B) Views of the EME binding site WT RACK1 (A) and S278E RACK1 (B) 80S reconstructions (rotated state shown). In S278E RACK1, an unidentified density connects G961 of the 18S rRNA with the E-site tRNA (asterisk). EME modeling based on PDB: 3J7A (Wong et al., 2014). (C) 35S-Met/Cys-labeling gels (top panel) and western blot analysis (bottom panels) of cells treated with the indicated concentrations puromycin (Puro). Representative of 3 independent biological replicates. (D) Top: schematic of control or poly(A) RQC reporters, with 2A protease and linker sites indicated. Bottom: densitometry-based quantification of GFP and RFP from western blot analysis of cells transfected with RQC reporters, presented as the RFP:GFP ratio. n = 3; no rescue **p = 0.002, WT rescue **p = 0.006, S278E rescue *p = 0.037; unpaired t test between control and poly(A) reporter. The numeric difference in ratio between each reporter is also shown. (E) Fluorescence intensity measurements of GFP or RFP (reported as arbitrary units) in cells transfected with RQC reporters, presented as violin plots. n = number of…

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  Figure 4.. A negatively charged RACK1 loop broadly enables eIF4A-independent translation (A and B) Fluorescence intensity measurements of GFP and RFP (reported as arbitrary units) in cells transfected with control (Ctrl) or poly(A) RQC reporters as in Figure 3E. Each cell is presented as an individual data point. n = number of fluorescent cells analyzed over 3 independent biological replicates. The whole dataset is shown in (A). The zoomed dataset in (B) highlights the large population of cells in S278E RACK1 rescue lines that express RFP but little GFP. (C and D) 35S-Met/Cys-labeling gels (top panel) and western blot analysis (bottom panels) of cells treated with the indicated concentrations of hippuristanol (Hipp; C) or silvestrol (Silv; D). Red bars/arrows highlight examples of proteins whose synthesis is repressed by inhibitors. Green arrows highlight examples of proteins whose synthesis is sustained. Representative of 3 independent biological replicates. See also Figure S6.

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  Figure 5.. The 40S head is displaced in S278E RACK1-containing ribosomes (A) Rigid-body fits of the human 80S ribosome in the nonrotated state (PDB: 4UG0) show agreement in the 60S and 40S body for both WT and S278E RACK1 reconstructions. (B) In contrast, the fitting is inconsistent at the 40S head between the two reconstructions (WT, gray; S278E, purple). Arrows indicate the direction of S278E 40S head displacement toward the 60S. (C) Closeup views of the RACK1-eS17 interface in WT (left) and S278E (right) reconstructions of nonrotated 80S particles. eS17 contains a connecting helix between the 40S body and the 40S head, which is less pronounced in the S278E reconstruction (asterisk). (D) Reconstruction of 40S particles isolated from S278E RACK1 purifications (left). Zoomed-in view of the latch separating the 40S head and body. The distance between Q179 of uS3 and G610 of the 18S rRNA is indicated and consistent with the 40S latch in the closed conformation. (E) Reconstruction of S278E 40S particles shows agreement with the rigid-body fit of the 40S ribosome bound to HCV IRES (PDB: 5A2Q). (F) Overlaid models of IRES-bound 40S subunits are generally superimposable (HCV, orange, PDB: 5A2Q;…