40S ribosome remodeling triggers 18S rRNA U-tailing and DIS3L2-dependent decay.

Eliminating defective ribosomes through quality control is essential for accurate protein synthesis. However, the mechanisms that commit defective ribosomal subunits to decay remain poorly defined. Here, we identify a tandem mechanism in which ubiquitin-dependent ribosome remodeling and 18S rRNA uridylation lead to 40S ribosomal subunit decay. Specifically, we use an in vitro reconstitution system to show that the atypical kinase RIOK3 remodels 40S ribosomal subunits, thereby exposing the 3' end of 18S rRNA. Nanopore direct RNA sequencing reveals that this remodeling event promotes oligo-uridylation, generating uridylated 18S rRNA decay intermediates. Uridylated 18S rRNA is further degraded by the 3'-5' exoribonuclease DIS3L2. Moreover, DIS3L2-mediated exoribonucleolytic cleavage triggers endoribonucleolytic decay of the 18S rRNA, amplifying turnover. Together, our findings define a stepwise mechanism in which ribosome remodeling and RNA tailing commit defective 40S subunits to elimination, establishing a mechanistic framework for ribosome surveillance in mammalian cells.