A Study of Mechanism of RACK1-dependent ZAKα activation at stalled and collided ribosomes. Vind et al.

Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38 and JNK-activating MAP3 kinase ZAKa senses translational impairment. Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKa dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKa via direct interactions with the ribosomal proteins RACK1 and RPS27 as well as 18S rRNA helix-26. In this conformation, the RNA-binding S (sensing) and C-terminal domain of ZAKa span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAKa off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKa to slow-elongating, stalled and collided ribosomes is associated with sequestration of the SAM domain on RACK1, allowing for transient ZAKa dimerization, activation loop trans-autophosphorylation and RSR activation. Our findings highlight how ZAKa senses both stalled and collided ribosomes through overlapping mechanisms.