Abstract
The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs.