Schematic of Rio1-mediated QC step monitoring 18S rRNA cleavage. (A) QC mechanism regulated by Rio1 ensures only ribosomes with precise 18S rRNA cleavage are licensed to translate. The red loop on the ribosomes next to Nob1 indicates ITS1 in pre-18S rRNA. Failure to pass this QC step results in turnover of these 80S-like intermediates. (B) Bypassing QC via weakly binding Pno1 mutations leads to the release of ribosomes containing uncleaved 20S pre-rRNA or miscleaved 18S rRNA into the translating pool, where they cause errors in translation (20S pre-rRNA–containing ribosomes; Parker et al., 2019) or ribosome collisions (miscleaved 18S rRNA-containing ribosomes—not shown; Parker et al., 2022Preprint). (C) Structure of the human pre-40S ribosomal subunit (3′-end of 18S rRNA is gray and RPs are removed for clarity) bound by PNO1 (purple) and NOB1 (green and black; PDB accession no. 6ZXE; Ameismeier et al., 2020). Colored spheres indicate residues in PNO1 or NOB1 that are mutated in cancer and are predicted to bypass QC. Pink spheres indicate nonsense mutations in NOB1 (C234, Y334, and Q348) and black indicates the section of NOB1 truncated after S325 (homologous to yeast Nob1_1-363; Parker et al., 2019). Mutations in PNO1 include PNO1_K186/K189/K191/F192 (homolog to yeast Pno1_KKK/F; yellow) and cancer mutations PNO1_T190N (homolog to yeast Pno1_T212N; cyan) and PNO1_R84I, P87Q, A184T (orange; TCGA www.cancer.gov/tcga, cBioPortal www.cbioportal.org).