Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Abstract

Eukaryotic circadian clocks employ autoregulatory negative feedback loops to control daily rhythms. In the filamentous fungus Neurospora, FRQ, FRH, WC-1, and WC-2 are the core components of the circadian negative feedback loop. To close the transcription-based negative feedback loop, the FRQ-FRH complex inhibits the activity of the WC complex in the nucleus by promoting the casein kinases-mediated WC phosphorylation. Despite its essential role in the nucleus, most FRQ is found in the cytoplasm. In this study, we mapped the FRQ regions that are important for its cellular localization. We show that the C-terminal part of FRQ, particularly the FRQ-FRH interaction domain, plays a major role in controlling FRQ localization. Both the mutation of the FRQ-FRH interaction domain and the down-regulation of FRH result in the nuclear enrichment of FRQ, suggesting that FRH regulates FRQ localization via a physical interaction. To study the role of FRQ phosphorylation, we examined the FRQ localization in wild-type as well as an array of FRQ kinase, FRQ phosphatase, and FRQ phosphorylation site mutants. Collectively, our results suggest that FRQ phosphorylation does not play a significant role in regulating its cellular localization. Instead, we find that phosphorylation of FRQ inhibits its transcriptional repressor activity in the circadian negative feedback loop. Such an effect is achieved by inhibiting the ability of FRQ to interact with WCC and casein kinase 1a. Our results indicate that the rhythmic FRQ phosphorylation profile observed is an important part of the negative feedback mechanism that drives robust circadian gene expression.The eukaryotic circadian oscillators are comprised of autoregulatory negative feedback loops (1-5). Despite the evolutionary distance between the filamentous fungus Neurospora crassa and higher eukaryotes, their circadian oscillator mechanisms share remarkable similarities (6 -8). In N. crassa, two PAS (PER-ARNT-SIM) domain-containing transcription factors, WC-1 and WC-2, form a complex (WCC) 2 that activates the transcription of the frq gene by binding to its promoter (9 -13). FRQ protein binds FRH to form the FRQ-FRH complex (FFC), which acts as the negative element in the circadian negative feedback loop (14 -17). To close the circadian negative feedback loop, FFC decreases frq mRNA-levels by promoting frq mRNA degradation and by inhibiting frq transcription. To repress frq transcription, FFC inhibits WCC activity by recruiting casein kinase 1a (CK-1a) and CKII to phosphorylate the WC proteins, resulting in a decrease of WCC DNA binding activity and an increase of WCC nuclear export (9, 10, 14 -24). Like the animal PER proteins, FRQ is progressively phosphorylated upon its synthesis and becomes extensively phosphorylated before its disappearance, resulting in robust oscillations of its level and phosphorylation profile (25). Under normal physiological conditions, FRQ is phosphorylated by CK-1a, CKII, and PKA (21,22,(25)(26)(27)(28). On the other hand, FRQ is also dephosphorylated and st...

The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa

“…Moreover, CK1a is activated by recruitment to FRQ. In this way, a single molecule of bound CK1a could phosphorylate in cis a large number of sites that are distributed all over the intrinsically disordered polypeptide chain of FRQ and also WCC that is recruited to the FFC 16,17,29 .…”

“…FRH is essential for the viability of Neurospora owing to its function in RNA metabolism 14 . In addition, FRH has been implicated in a variety of non-essential, clock-related functions: FRH is crucial for cytoplasmic localization and turnover of FRQ 15,16 , and also for the interaction of FRQ with the circadian transcription activator White Collar Complex (WCC) 15,17 . Furthermore, it was reported that FRH mediates turnover of frq RNA by committing the messenger for degradation by the exosome 14 .…”

The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA

“…Interestingly, this dual effect of ZTL on GI is reminiscent of the relationship between FREQUENCY (FRQ) and an RNA helicase (FRH) in the Neurospora clock. The formation of a FRQ-FRH complex (FCC) helps stabilize and retain FRQ in the cytosol, with increased FRQ nuclear levels when the interaction is disrupted or FRH levels are low (Cha et al, 2011). Conversely, the mammalian clock component mCRY helps retain the nucleocytoplasmic protein mPER2 in the nucleus and away from ubiquitylation and degradation in the cytosol (Yagita et al, 2002).…”

The Genetics of Circadian Rhythms in Neurospora

“…Despite the presence of the nuclear localization signal, most FRQ protein is cytoplasmic, suggesting that there is an active process to export FRQ from the nucleus. Using FRQ deletion mutants, the C-terminus of FRQ that includes the FRQ/FRQ-interacting RNA helicase (FRH) interaction domain was found to play an important role for cytoplasmic localization of FRQ (Cha et al ., 2011). Other domains in FRQ include a casein kinase I (CK1) interaction domain located in the middle of the protein (Guo et al ., 2010) and two PEST domains that function in FRQ turnover (Görl et al ., 2001).…”