| dc.description.abstract | At least half of proteins cycling in abundance under control of the circadian clock in eukaryotic cells are synthesized from non-cycling mRNAs. These data suggested that the clock controls posttranscriptional events, including mRNA translation; however, the mechanisms underlying this regulation were not known. We, and other labs, discovered that the circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the peak in inhibitory eIF2α phosphorylation (P-eIF2α) levels occurs during the subjective day. The activity of the N. crassa eIF2α kinase, CPC-3, was shown to be necessary for rhythmic P-eIF2α accumulation. However, it was not known if CPC-3 activity is sufficient for rhythmic P-eIF2α, or if other factors are involved. In this study, I tested the hypothesis that rhythmic eIF2⍺ activity also requires dephosphorylation of P-eIF2⍺ at night by phosphatases. In support of this hypothesis, I demonstrated that mutation of N. crassa PPP1, a homolog of the yeast eIF2⍺ phosphatase GLC7, leads to high and arrhythmic P-eIF2⍺ levels, while maintaining core circadian oscillator function. Furthermore, PPP1 levels are clock-controlled, peaking at dusk, and rhythmic PPP1 levels were shown to be necessary for rhythmic P-eIF2⍺ accumulation. I discovered that eIF2γ, a component of the eIF2 complex, functions to recruit PPP1 to dephosphorylate eIF2⍺. Thus, in addition to the activity of CPC-3 kinase, rhythmic P-eIF2α requires dephosphorylation by PPP1 phosphatase at night. To determine the impact of rhythmic eIF2α activity on mRNA translation, ribosome profiling, in parallel with RNA-seq, was carried out. In collaboration with Dr. Kathrina Castillo, I found that the N. crassa clock regulates the translation of 1328 mRNAs, of which 404 were translated arrhythmically in strains that lacked CPC-3 (∆cpc-3) or had a constitutively active CPC-3 (cpc-3c). These data revealed that rhythmic eIF2α activity regulates translation of specific, rather than all, mRNAs. Experiments are in progress to investigate the mechanisms of rhythmic P-eIF2α regulation of these targeted mRNAs. Together, these data show how the circadian clock regulates protein production by controlling the activity of a central regulator of translation through the temporal coordination of phosphorylation and dephosphorylation events. | en |
