Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a series of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide, and targeting of the mRNA for decay (No Go Decay or NGD). Using a reverse genetic screen in yeast, we identify Cue2 as the conserved endonuclease that is recruited to stalled ribosomes to promote NGD. Ribosome profiling and biochemistry provide strong evidence that Cue2 cleaves mRNA within the A site of the colliding ribosome. We demonstrate that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay and Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway. Finally, we show that the Cue2-dependent pathway becomes a major contributor to NGD in cells depleted of factors required for the resolution of stalled ribosome complexes. Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells.
