RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation.

The initiation-specific ribosome-associated quality control pathway (iRQC) is activated when translation initiation complexes fail to transition to elongation-competent 80S ribosomes. Upon iRQC activation, RNF10 ubiquitylates the 40S proteins uS3 and uS5, which leads to 40S decay. How iRQC is activated in the absence of pharmacological translation inhibitors and what mechanisms govern iRQC capacity and activity remain unanswered questions. Here, we demonstrate that altering 60S:40S stoichiometry by disrupting 60S biogenesis triggers iRQC activation and 40S decay. Depleting the critical scanning helicase eIF4A1 impairs 40S ubiquitylation and degradation, indicating mRNA engagement is required for iRQC. We show that amino acid starvation conditions also stimulate iRQC-dependent 40S decay. We identify RIOK3 as a crucial iRQC factor that interacts with ubiquitylated 40S subunits to mediate degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC pathway activation, establishing a feedforward mechanism that regulates iRQC capacity and subsequent 40S decay.