A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa

Abstract

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identi®ed Neurospora homologs of casein kinase 1e. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes signi®cantly increased protein stability. A strain harboring the mutant frqDPEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frqDPEST-1 RNA and FRQDPEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components. Keywords: casein kinase 1e/circadian/frequency/ Neurospora/PEST IntroductionMost organisms use circadian clocks to regulate physiological and behavioral activities in a time-of-day speci®c manner. Circadian clocks are cell-autonomous and mediate rhythmic expression of a broad variety of clockcontrolled genes with a periodicity of approximately one day even in the absence of environmental cues. Circadian clocks are entrainable by exogenous stimuli (zeitgebers) such as light, temperature and nutrition. Entrainment synchronizes the endogenous oscillations with the exogenous time and allows behavioral¯exibility. On the molecular level circadian oscillations are supported by interconnected transcriptional±translational feedback loops of considerable complexity. The interconnected loops seem to function in a similar manner in fungi,¯ies and mammals (Dunlap et al., 1999;Young, 2000;Albrecht, 2001;Reppert and Weaver, 2001;Williams and Sehgal, 2001).The circadian system of Drosophila melanogaster is well characterized on the molecular level. Drosophila CLOCK (CLK) and CYCLE (CYC) are Per-Arnt-Sim-like (PAS) domain-containing transcription factors that control transcription of period (per) and timeless (tim) RNA (Allada et al., 1998;Darlington et al., 1998;Rutila et al., 1998;McDonald et al., 2001;Wang et al., 2001). When PER and TIM proteins are synthesized they form a complex that enters the nucleus and represses transcription of their own genes by inactivating CLK±CYC (Hardin et al., 1990;Saez and Young, 1996;Lee et al., 1998Lee et al., , 1999. During the course of a day PER and TIM are degraded, which again allows synthesis of per and tim RNA by CLK±CYC (Young, 2000;Williams and Sehgal, 2001). The length of a circadian period is affected by the kinetics of nuclear entry and degradation of PER±TIM. TIM is phosphorylated by SHAGGY, which regulates nuclear translocation of the PER±TIM heterodimer (Martinek et al., 2001). The casein kinase 1e (CK-1e) homolog DOUBLETIME (DBT) triggers degradation of PER via phosphorylation and thereby regulates period length Rothen¯uh et al....

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

“…When protein synthesis was inhibited with cycloheximide, FRQ in complex with the FLAG FRH (followed by FLAG-IP) was degraded with a half-time of 3.5 h (Fig. 2c), which corresponds to the previously reported degradation kinetics of FRQ 6,23 . FRQ in complex with Walker A and DEAD box mutants of FLAG FRH was degraded significantly faster (Fig.…”

“…Fourhundred micrograms of N. crassa and 100 mg of S. cerevisiae extract were analysed by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare; 0.45 mm, 10 Â 150 cm) in 150 mM glycine, 20 mM Tris, 20% (v/v) methanol and 0.08% (w/v) SDS at 200 mA for 2.5 h. The membrane was blocked with TBS and 5% milk (pH 7.4) before incubation with antibodies 6,29 . Antibodies against FRQ were used 1:20 diluted in 5% milk and CK1a antibodies at a dilution of 1:500 in 5% milk 6,23 . An FRH (1:350 in 5% milk) antibody was raised in rabbits against a peptide corresponding to amino-acid residues 2-18 (Pineda, Berlin).…”

“…FRQ interacts with CK1a 6 and forms a tight complex with FRQ-interacting RNA helicase (FRH), an essential DEAD box-containing RNA helicase 7 . DEAD box-containing RNA helicases comprise a large superfamily of ATPases whose functions are largely unknown 8,9 .…”

Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

“…We estimate that ∼40%-50% of WC-1 is degraded through this process. Phosphorylation-dependent protein degradation has been demonstrated for FRQ in Neurospora, a critical process regulating the circadian clock (Liu et al 2000;Gorl et al 2001;Yang et al 2002Yang et al , 2004He et al 2003).…”

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

“…Previous studies have demonstrated that phosphorylation of circadian clock proteins is an essential posttranscriptional mechanism in the regulation of circadian clocks (Dunlap 1999;Young and Kay 2001). Several kinases, including casein kinase I (CKI) and CKII, have been shown to phosphorylate and regulate the key clock components in eukaryotic systems Sugano et al 1999;Lowrey et al 2000;Gorl et al 2001;Yang et al 2002).FREQUENCY (FRQ), WHITE COLLAR-1 (WC-1), and WC-2 proteins are three key components in the Neurospora frq-wc-based circadian oscillator . In addition to being the circadian blue light photoreceptor (Froehlich et al 2002;He et al 2002), in the dark, WC-1 forms a heterodimeric complex with WC-2 through their PAS domains and activate frq transcription by binding its promoter (Crosthwaite et al 1997;Talora et al 1999;Cheng et al 2001bCheng et al , 2002Cheng et al , 2003Froehlich et al 2003).…”