Yeast Rai1p and the RAC complex (Ssz1p/Zuo1p) modulate uORF-mediated GCN4 translational control under stress conditions.

Translation reinitiation (REI) is one of the most important gene-specific regulatory mechanisms by which eukaryotic cells influence expression of main translons, for example during highly conserved integrated stress response (ISR). In S. cerevisiae, expression of the key stress response gene, GCN4, is controlled by an intricate interplay among four short upstream translons (uTranslons, formerly uORFs), resulting in high or low levels of REI at GCN4 depending on the growth conditions. Under nutrient rich conditions, GCN4 expression is repressed, but upon amino acid starvation, it is derepressed, despite of a general translational shut down. Capitalizing on our screening reporter system, we identified three new factors influencing efficiency of REI after translation of GCN4 uTranslons: Rai1p (an RNA quality control and processing factor), and Ssz1p and Zuo1p (members of the Ribosome Associated Complex [RAC]). Importantly, we showed that depletion of these factors deregulated derepression of Gcn4p synthesis under starvation. Furthermore, we found that similar to RAC, Rai1p associates with 40S subunits and actively translating ribosomes. We also explored interactomes of these proteins. Collectively, we present three previously unknown factors that co-regulate stress response to amino acid starvation in the budding yeast by unique mechanisms.