Axel Diernfellner

Circadian conformational change of the Neurospora clock protein FREQUENCY triggered by clustered hyperphosphorylation of a basic domain.

In the course of a day, the Neurospora clock protein FREQUENCY (FRQ) is progressively phosphorylated at up to 113 sites and eventually degraded. Phosphorylation and degradation are crucial for circadian time keeping, but it is not known how phosphorylation of a large number of sites correlates with circadian degradation of FRQ. We show that two amphipathic motifs in FRQ interact over a long distance, bringing the positively charged N-terminal portion in spatial proximity to the negatively charged middle and C-terminal portion of FRQ. The interaction is essential for the recruitment of casein kinase 1a (CK1a) into a stable complex with FRQ. FRQ-bound CK1a progressively phosphorylates the positively charged N-terminal domain of FRQ at up to 46 nonconsensus sites, triggering a conformational change, presumably by electrostatic repulsion, that commits the protein for degradation via the PEST1 signal in the negatively charged central portion of FRQ.

A Global Circadian Repressor Controls Antiphasic Expression of Metabolic Genes in Neurospora

The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes.

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion.

Long and short isoforms of Neurospora clock protein FRQ support temperature compensated circadian rhythms

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l‐FRQ vs. s‐FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l‐FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l‐FRQ or s‐FRQ support short and long temperature‐compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine‐tune period length in response to ambient temperature.

Light input and processing in the circadian clock of Neurospora

Circadian clocks are endogenous oscillators that use zeitgebers as environmental cues to synchronise with the exogenous day–night cycle. The role of light as a zeitgeber has been investigated intensively to date. In Neurospora crassa the transcription factor White Collar Complex (WCC) is directly activated by light, which resets the clock. In addition, a hierarchical cascade of transcription factors activates the light‐induced expression of hundreds of genes. Disturbance of the clock during the day through changes in light intensity should be prevented to ensure efficient synchronisation. This can be achieved by desensitisation to the ambient light (photoadaptation). Photoadaptation in Neurospora is dependent on the blue light receptor Vivid (VVD), which accumulates immediately after light activation and rapidly silences the expression of WCC‐dependent genes. Recent studies have elucidated the molecular mechanism of VVD‐mediated photoadaptation. Here we review the increasing knowledge about light‐dependent gene expression and photoadaptation in Neurospora and discuss their relevance for synchronisation of the circadian clock.

Phosphorylation modulates rapid nucleocytoplasmic shuttling and cytoplasmic accumulation of Neurospora clock protein FRQ on a circadian time scale

The Neurospora clock protein FREQUENCY (FRQ) is an essential regulator of the circadian transcription factor WHITE COLLAR COMPLEX (WCC). In the course of a circadian period, the subcellular distribution of FRQ shifts from mainly nuclear to mainly cytosolic. This shift is crucial for coordinating the negative and positive limbs of the clock. We show that the subcellular redistribution of FRQ on a circadian time scale is governed by rapid, noncircadian cycles of nuclear import and export. The rate of nuclear import of newly synthesized FRQ is progressively reduced in a phosphorylation-dependent manner, leading to an increase in the steady-state level of cytoplasmic FRQ. The long-period frq(7) mutant displays reduced kinetics of FRQ(7) protein phosphorylation and a prolonged accumulation in the nucleus. We present a mathematical model that describes the cytoplasmic accumulation of wild-type and mutant FRQ on a circadian time scale on the basis of frequency-modulated rapid nucleocytoplasmic shuttling cycles.

Phosphorylations: making the Neurospora crassa circadian clock tick

Various post‐translational modifications have been identified that play a role in the function of circadian clocks. Among these, phosphorylation has been investigated extensively. It was shown that phosphorylation influences half‐life, subcellular localisation, transcriptional activity and conformation of clock components over the course of a circadian day. Recent observations also indicate that time‐of‐day specific sequential phosphorylation of the Neurospora crassa clock protein FREQUENCY is crucial for measuring time and thus for establishing a robust circadian rhythm. The circadian clock of Neurospora is one of the best‐investigated molecular clocks to date. In this review, we summarise the data on what is known so far about the role of phosphorylation of proteins involved in the Neurospora circadian clock.

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

The Neurospora clock protein FRQ forms a complex with casein kinase 1a (CK1a) and FRH, a DEAD box-containing RNA helicase with a clock-independent essential function in RNA metabolism. In the course of a circadian period, FRQ is progressively hyperphosphorylated and eventually degraded. Timed hyperphosphorylation of FRQ is crucial for timekeeping of the clock. Here we show that the ATPase activity of FRH attenuates the kinetics of CK1a-mediated hyperphosphorylation of FRQ. Hyperphosphorylation of FRQ is strictly dependent on site-specific recruitment of a CK1a molecule that is activated upon binding. The FRH ATPase cycle regulates the access of CK1a to phosphorylation sites in FRQ in cis, suggesting that FRH is an ATP-dependent remodelling factor acting on the protein complex. We show that the affinity of CK1a for FRQ decreases with increasing FRQ phosphorylation, suggesting functional inactivation of FRQ in the negative feedback loop of the circadian clock before and independent of its degradation.

The Neurospora photoreceptor VIVID exerts negative and positive control on light sensing to achieve adaptation.

The light response in Neurospora is mediated by the photoreceptor and circadian transcription factor White Collar Complex (WCC). The expression rate of the WCC target genes adapts in daylight and remains refractory to moonlight, despite the extraordinary light sensitivity of the WCC. To explain this photoadaptation, feedback inhibition by the WCC interaction partner VIVID (VVD) has been invoked. Here we show through data‐driven mathematical modeling that VVD allows Neurospora to detect relative changes in light intensity. To achieve this behavior, VVD acts as an inhibitor of WCC‐driven gene expression and, at the same time, as a positive regulator that maintains the responsiveness of the photosystem. Our data indicate that this paradoxical function is realized by a futile cycle that involves the light‐induced sequestration of active WCC by VVD and the replenishment of the activatable WCC pool through the decay of the photoactivated state. Our quantitative study uncovers a novel network motif for achieving sensory adaptation and defines a core input module of the circadian clock in Neurospora.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.