Site-specific K63 ubiquitinomics reveals stabilization of ribosomes in response to oxidative stress

Reprograming of protein synthesis is an essential cellular process to tolerate and resist to stressing conditions. A variety of mechanisms are known to regulate translation at initiation but cells can also control proteins synthesis after the initiation checkpoint. We previously showed that K63 ubiquitin can modify ribosome proteins in response to oxidative stress. However, the mechanism by how K63 ubiquitin impacts ribosome function is entirely unknown. Here we characterized > 1000 K63 ubiquitin sites in the yeast Saccharomyces cerevisiae by mass spectrometry, and showed that many sites clustered at the head of the 40S subunit in response to H2O2. Moreover, ribosomes lacking K63 ubiquitin were depleted in proteins from the translation initiation factor eIF3, particularly Tif35 (eIF3g), which impacted ribosome stability, and protein production. Our results provided new insights on the role of K63 ubiquitin in regulating the resistance to oxidative stress via a post-initiation control of translation.