Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGD^RQC+) or RQC uncoupled (NGD^RQC−). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGD^RQC− but not for NGD^RQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGD^RQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGD^RQC−), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGD^RQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.