RPS15 mutant proteins alter ribosomal activity at different levels. (A) Global view of RPS15 structure in the context of the ribosome, showing the first amino acids of its C-terminal region extending into the ribosomal decoding center. The figure was generated with UCSF Chimera 1.11.2 (https://www.cgl.ucsf.edu/chimera) by using the structure of the human wild-type ribosome (PDB 5AJ0). The RPS15 protein is shown in a ribbon representation, whereas RNAs are shown as balls and sticks. The rRNAs of the large ribosomal subunit (28S, 5.8S, and 5S) are represented in gray, the rRNA of the small ribosomal subunit (18S) is in yellow, the tRNA is in blue, the mRNA is in cyan, and RPS15 protein (amino acids 12-131) is in red. (B) ³⁵S-Met/Cys incorporation at the different times in protein precipitates from GFP-RPS15–expressing (wild-type or mutants) HEK293T cells. Signal intensities of autoradiography analyses were quantified, and mean values from ≥3 independent experiments are represented. Error bars indicate SEM. *P < .033, **P < .002, ***P < .001, two-way ANOVA. (C) Comparative ³⁵S-Met/Cys incorporation between GFP-RPS15^WT, GFP-RPS15^P131S, and GFP-RPS15^S138F cell lines. A representative autoradiography image and the associated RPS15 and β-actin immunoblots of 3 independent experiments are shown. (D-F) Dual-luciferase assays were performed to quantify cap-independent initiation (D), amino acid misincorporation (E), and stop codon read-through (F) during ribosomal translation in GFP-RPS15–expressing (wild-type or mutants) HEK293T cells. Firefly/renilla or renilla/firefly ratios were set to 1.0 in the case of GFP-RPS15^WT–expressing cells (dark blue bars). Each bar represents the mean of 3 independent determinations + SEM. In (D-F), *P < .033, **P < .002, ***P < .001, one- or two-way ANOVA.