ZAKα is a sensor of mRNA stasis at the ribosomal exit channel

Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38 and JNK-activating MAP3 kinase ZAKα senses translational impairment. Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKα dynamically monitors the mRNA exit channel of elongating ribosomes for mRNA stasis. This is accomplished by ZAKα via its direct interactions with the ribosomal proteins RACK1 and RPS27 as well as with the 18S rRNA helix-26. In this conformation, four mRNA-binding peptides in ZAKα span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAKα off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKα to slow-elongating, stalled and collided ribosomes is associated with sequestration of the inhibitory SAM domain on RACK1, allowing for transient ZAKα dimerization, activation loop trans-autophosphorylation and RSR activation. We propose that compromised ribosome processivity constitutes a common ribotoxic stress signal and that ZAKα is a ribosome collision-agnostic sensor of such perturbations.