Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding

Figures

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  Figure 1.. Generation of RPS3 mRNA binding mutant cell lines. (A) The structure of human RPS3 [Protein Data Bank (PDB) ID: 4V6X] was aligned to yeast RPS3 from the 48S PIC [PDB ID: 3J81] using PyMol (shown in cartoon format). Human RPS3 is colored in green, yeast RPS3 in turquoise, mRNA backbone in orange and sidechains are colored in light blue with blue and red. Residues near the mRNA are highlighted in grey or red. mRNA position + 6 is highlighted in black. (B) Immuno-blot analysis of the RPS3 mutant clones, normalized to protein concentration measurement, using an anti-RPS3 antibody that recognizes amino acid sequence 100–161(upper panel) or amino acid sequence 193–243 (lower panel). (C–F) Polysomal profiling of RPS3 mutant clones. Cell lysates of RPS3+/− (C), R116D/− (D), R146D/− (E) and K148D/− (F) were subjected to sucrose gradient sedimentation followed by fraction collection. Representative profiles are shown. G. Quantification of polysomes/monosome (P/M) and 40S/60S ratios from the polysome profiling, N = 4. (H) Free, 40S, 60S and 80S fractions were subjected to western blotting using anti-RPS3 and anti-RPS5. Percentages of association were quantified, N = 2. Representative…

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  Figure 2.. The impact of RPS3 mutations on ribosomal composition. (A)Analysis of the mRNA levels of RPS3 relative to GAPDH in RPS3+/+, RPS3+/− and R116D/- was measured by qPCR. Relative expression values normalized to the values in RPS3+/+, N = 3. (B) Growth curve of RPS3+/+ and the RPS3 mRNA binding mutant clones. An equal number of RPS+/+, RPS3+/− and mutated RPS3 cells were seeded, and proliferation was measured using luminescent cell viability assay at indicated time points. Luminescence values were normalized to the values of day 1, N = 4. (C)Polysome profiling representations of RPS3+/+ and RPS3+/− cells. Representative profiles are shown. (D)Quantification of RPS3 relative to RPS14 in 80S fractions of RPS3+/+ and RPS3+/− by western blotting. The relative values are normalized to values of RPS3+/+, N = 3. The graphs represent the means ± the SE. Asterisks denote a statistically significant difference (*P< 0.05, **P< 0.01).

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  Figure 3.. The effect of the mutations in RPS3 mRNA binding residues on cap-proximal initiation and start codon fidelity. (A–E) RPS3 mutant clones were co-transfected with a GFP reporter plasmid driven by a non-optimal Kozak AUG context (A, B) (N ≥ 3) or TISU (C, D) (N ≥ 2) with short 5′UTR (16 nt or 10 nt) or long 5′UTR (94 nt) and a Kozak context (E) (N = 5) together with a Firefly-luciferase as normalizing control. Schematic representations of the GFP reporter gene are shown on the top of each panel. US and DS denote the upstream and downstream translation initiation sites, respectively. The arrow indicates the position of the TSS. The expected translation initiation sites are indicated. Cells were harvested 24 h after transfection and subjected to western blotting using an anti-GFP antibody. Representative immuno-blots are shown. Quantified results are shown in the graphs in which the relative intensity of the upstream translation site is presented by dark bars (SE in black) and the downstream translation site by light bars (SE in grey). The overall translation of RPS3+/- cells was set to one. Grey and black asterisks on the graphs denote a statistically significant difference in…

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  Figure 4.. Translatome analysis RPS3 of mRNA binding mutant clones reveals common regulation of scanning start site selection. (A) Venn diagrams of the Downregulated and Upregulated genes in each sample R116D/–, R146D/– or K148D/– compared to the RPS3+/– sample (1.4-fold change). (B) Graphs representing the frequency of different AUG contexts of the CDS with alternating –3 and +4 positions among the Unchanged, Downregulated (Down) and Upregulated (Up) genes in each of the mutants. Strong Kozak context is RNNAUGG, partial Kozak is YNNAUGG and RNNAUGH and weak context is YNNAUGH. (C, D) Boxplots presenting the 5′UTR (C) and CDS (D) length distribution among the unchanged, downregulated (Down) and upregulated (Up) genes common to all mutants. Asterisks denote a statistically significant difference (*P < 0.05, **P < 0.01, ***P< 0.005, ****P < 0.001).

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  Figure 5.. RPS3 mRNA binding residues are critical for NSP1-mediated translation inhibition. (A)A Firefly reporter gene (with non-viral 5’UTR) was transfected into RPS3+/– and mutant cells in the presence of either GFP or NSP1 expression plasmids as indicated. N ≥ 6. (B) A Renilla luciferase reporter gene driven by the sub-genomic 5’UTR of SARS-CoV-2 was transfected into RPS3+/– and mutant cells in the presence of either GFP or NSP1 expression plasmids as indicated. N ≥ 7. (C)Polysome profiles followed by a western blot analysis of RPS3+/– and R116D/– cells transfected with HA-NSP1 plasmid. The indicated fractions were tested with antibodies against NSP1 (anti-HA), RPS14, and RPL17. The graph represents the distribution of NSP1 in fractions in the different strains, N = 2. Representative profiles and immune-blots are shown. (D) Relative level of poly A + mRNA isolated from RPS3+/– and R116D/– cells transfected with GFP or NSP1 expressing plasmids, N = 4. The graphs represent the means ± the SE. Asterisks denote statistically significant difference (*P< 0.05, **P< 0.01, ****P < 0.001).

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  Figure 6.. Identification of an RPS3RS, an RPS3-dependent inhibitory motif in SARS-CoV-2 sub-genomic 5’UTR. (A)The activity of the SARS-CoV-2 sub genomic 5’UTR Renilla reporter relative to normalizing Firefly reporter gene in RPS3+/− and mutant RPS3 strains. All the values are relative to RPS3+/−, N = 8. (B) The sequence of the sub-genomic 5’UTR 72 nucleotides of SARS-CoV-2. The RPS3RS element is colored with the CUG in red and the downstream nucleotides that are part of the ‘TRS element’ in yellow. (C)The effect of CUG substitution to CAG (CAG) and CUG DS 5 nucleotides-AAACG substitution to TTTGC (DS mut) on the normalized expression of SARS-CoV-2 5’UTR- Renilla in RPS3+/− and mutant strains. CON (control) refers to SARS-CoV-2 5’UTR- Renilla plasmid without mutation. All the values normalized to the values of RPS3+/- with the control plasmid, N ≥ 4. (D)The effect of RPS3RS (RPS3 regulatory sequence) insertion in two positions in the 5’UTR of Firefly reporter gene on its normalized expression (relative to expression of Renilla reporter gene). The RPS3RS was inserted 4 and 9 nucleotides upstream of the AUG start codon (RPS3RS −4 and RPS3RS −9, respectively). CON (control) refers to the…