Abstract
Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock's positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.
Highlights
The Neurospora circadian oscillator is a transcription–translation feedback loop that is positively regulated by the White Collar Complex (WCC) transcription factors, which drive expression of the negative arm component Frequency (FRQ)
Placing NCU01019 under the nutrient-responsive qa-2 promoter, we find that the long period length occurs at very low gene expression levels using 10À6 M quinic acid induction (Figure 1E)
By uncovering the identity and mode of action of PRD-2 and exploring the mechanism of two classical period mutants, prd-2 and upf1prd-6, we found a common basis in regulation of casein kinase I (CKI) levels, which are under tight control in the Neurospora clock (Figure 6)
Summary
The Neurospora circadian oscillator is a transcription–translation feedback loop that is positively regulated by the White Collar Complex (WCC) transcription factors, which drive expression of the negative arm component Frequency (FRQ). In this way the fungal core circadian oscillator shares a common regulatory architecture with the mammalian core clock. A unifying model emerges to explain the action of diverse period mutants, where the casein kinase I transcript is subject to complex regulation by NMD and an RNA-binding protein, PRD-2, to control its gene expression and maintain a normal circadian period
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