eL28 and RACK-1 are missing from the map, but exist in C. elegans. (A) Nt 445–513 of ES7L are poorly structured. The model is shown in colors, and the map density is in gray mesh. The approximate position of the missing segment of ES7L is indicated. In other organisms, this segment of ES7L binds eL28. (B) Conservation of ribosomal protein genes between C. elegans, D. melanogaster, and humans. The percent identity of each C. elegans ribosomal protein homolog is shown, with eL28 and RACK-1 highlighted. (C) mRNA expression of all genes (gray), ribosomal protein genes (blue), RACK-1 (green), and eL28 (red). mRNA expression was determined by Oxford Nanopore direct RNA-seq using published data (Viscardi and Arribere 2022). (D) mRNA translation of the indicated genes; same colors as C. mRNA translation was determined by ribosome footprint profiling (Ribo-seq) using published data (Stadler and Fire 2011). (E) eL28 conservation overlaid on eL28 binding site. eL28 was positioned in the ribosome using D. melanogaster as a model. eL28 protein model from AlphaFold (Varadi et al. 2022). Conservation data from consurf (Yariv et al. 2023), with more conserved residues in red and less conserved residues in blue. Note that ES7L would lay across the top of eL28, contacting the less-conserved regions of the protein. (F) Atomic model for RACK-1 binding in C. elegans, with map in gray mesh and model in colors. RACK-1 model from AlphaFold, with conservation colored as with eL28. Inset shows two salt bridges conserved in C. elegans that are known to be important for RACK-1::ribosome association in Saccharomyces cerevisiae. (G) Viability and developmental timing of the indicated strains. P-values are from Welch's t-test and are for comparison with wild-type (WT) animals; n.s., not significant. (H) unc-54(rareArg) reporter diagram and its expression in the indicated strains; the internal rareArg stretch stalls ribosomes and elicits mRNA decay (blue triangle). znf-598 is a positive control that is required for repression of the unc-54(rareArg) reporter; WT. (I) unc-54(Nonstop) reporter diagram and its expression in the indicated strains; the lack of an in-frame stop codon leads to ribosomal stalling at the 3′ end (triangle), eliciting mRNA repression, and causing low GFP levels. skih-2 is a positive control that is required for the repression of the unc-54(Nonstop) reporter.
