Sensing of individual stalled 80S ribosomes by Fap1 for non-functional rRNA turnover. Li et al

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself. Overall design: 20 ribosome profiling (ribo-seq) samples are included in the study. Ribo-seq samples were obtained from (1) total cell lysates (input) collected prior to purification, (2) MS2-purified ribosomes carrying exogenously encoded wild type or mutant rRNA, or (3) co-purified ribosomes from pull-downs of proteins of interest (Mag2, Fap1, or Yil161w). Input samples serve as controls that are compared against purified ribosomes. Each sample has a biological replicate.