Listerin-Dependent Nascent Protein Ubiquitination Relies on Ribosome Subunit Dissociation

Quality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes. •Nascent protein ubiquitination on stalled ribosomes has been reconstituted in vitro•Ubiquitination on separated 60S subunit is strongly preferred over intact 80S ribosome•Nascent chain inside a 60S subunit cues stable binding of ubiquitin ligase Listerin•Subunit dissociation by ribosome recycling factors enables ligase recruitment