a b s t r a c tThe RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5 0 untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.

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Rok1, a DEAD-box RNA helicase, is involved in rRNA processing and the control of cell cycle progression in Saccharomyces cerevisiae. Rok1 protein expression is cell cycle-regulated, declining at G1/S and increasing at G2. The downregulation of Rok1 expression in G1/S phase is mediated by the inhibitory action of two upstream open reading frames (uORFs) in the ROK1 5'-untranslated region (5'UTR). We identified Psk2 (PAS kinase), Skp1 (kinetochore protein) and Tub4 (γ-tubulin protein) as ROK1 5'UTR-interacting proteins using yeast three-hybrid system. A deletion analysis of PSK2 or inactivation of temperature-sensitive alleles of SKP1 and TUB4 revealed that Rok1 protein synthesis is repressed by Psk2 and Skp1. This repression appeared to be mediated through the ROK1 uORF1. In contrast, Tub4 plays a positive role in regulating Rok1 protein synthesis and likely after the uORF1-mediated inhibitory regulation. These results suggest that 5'UTR-interacting proteins, identified using three hybrid screening, are important for uORF-mediated regulation of Rok1 protein expression.