A new role for the DEAD-box RNA helicase DDX3 in co-translational protein quality control

DEAD-box RNA helicases are central players in RNA metabolism, however, their role in translation regulation is largely unexplored in parasitic protozoa. Here, we have investigated the role of DDX3 RNA helicase in ribosome-associated protein quality control in Leishmania. We show that ribosomes move more slowly and de novo polypeptide synthesis is reduced in cells lacking DDX3. In accordance with the slowing of ribosome speed, DDX3 depleted cells exhibit higher levels of ribosome-associated ubiquitination. Especially, ubiquitination of nascent polypeptides is enhanced upon DDX3 loss as determined by the isolation of ribosome-associated nascent chains modified either by HA-Ubiquitin or by endogenous ubiquitin using biotinylated-puromycin labeling. Consistent with increased co-translational ubiquitination, quantitative proteomics analysis revealed higher recruitment of E3 ubiquitin ligases and proteasomal components to DDX3 knockout ribosomes to eliminate aberrant nascent polypeptides. In addition, we show that cells lacking DDX3 accumulate cytosolic aggregates. This along with the higher recruitment of ribosome-associated chaperones and the improvement of translation by increasing HSP70 availability suggests that co-translational control of nascent polypeptides is impaired in the absence of DDX3. Altogether, these results highlight an important role for DDX3 in ribosome-associated quality control by reducing co-translational ubiquitination and proteotoxicity, hence allowing optimal ribosome movement and translation elongation.