Decoupling circadian clock protein turnover from circadian period determination

Larrondo, Luis F.; Olivares-Yañez, Consuelo; Baker, Christopher L.; Loros, Jennifer J.; Dunlap, Jay C.

doi:10.1126/science.1257277

Cited by 142 publications

(198 citation statements)

References 54 publications

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“…In a recent work the speed of the phosphorylation cascade affecting FRQ activity in the negative feedback was proposed as the key factor determining circadian period (15). Our data showing delayed phosphorylation of FRQ in DD in ras2 together with a longer period are in good accordance with this model.…”

Section: Discussionsupporting

confidence: 80%

“…The FRQ protein interacts with an RNA helicase (FRH) and the casein kinase 1a (9,14) and inhibits the activity of the WCC by facilitating its phosphorylation. FRQ undergoes several steps of phosphorylation that affect distinct functions of the protein (15)(16)(17). Shortly after its synthesis, low levels of hypophosphorylated FRQ forms are accumulated in the nucleus and are efficient in the negative feedback.…”

Section: Introductionmentioning

confidence: 99%

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The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa

Gyöngyösi

Szőke

Ella

et al. 2017

Journal of Biological Chemistry

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Accumulating evidence from both experimental and clinical investigations indicate a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and therefore is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in wild type (wt), in ras2 operation of the circadian clock was affected by glucose: compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Whereas glucose did not affect the subcellular distribution of FRQ in wt, highly elevated FRQ levels were detected in the nucleus in ras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-Br-cAMP partially rescued the circadian phenotype in vivo. We propose therefore that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

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Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa

Gyöngyösi

Szőke

Ella

et al. 2017

Journal of Biological Chemistry

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“…Phosphatase treatment (#P0753S, New England Biolabs) and coimmunoprecipitation using GammaBind G Sepharose beads (#17-0885-01, GE Healthcare Bio-Sciences) were as recommended by the manufacturer. In vitro translation of luc mRNA (67,68), in vivo luciferase assays (69), and statistical analysis of rhythmic data (15) were accomplished as previously described. Detailed methods can be found in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

Caster

Castillo

Sachs

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs.C ircadian rhythms are the outward manifestation of an endogenous clock mechanism common to nearly all eukaryotes. Depending on the organism and tissue, nearly half of an organism's expressed genes are under control of the circadian clock at the level of transcription (1-4). However, mounting evidence indicates a role for the clock in controlling posttranscriptional mechanisms (5), including translation initiation (6, 7), whereas clock control of translation elongation has not been investigated.The driver of circadian rhythms in Neurospora crassa is an autoregulatory molecular feedback loop composed of the negative elements FREQUENCY (FRQ) and FRQ RNA-interacting helicase (FRH), which inhibit the activity of the positive elements WHITE COLLAR-1 (WC-1) and WC-2 (8-11). WC-1 and WC-2 heterodimerize to form the white collar complex (WCC), which activates transcription of frequency (frq) (8,12,13) and activates transcription of a large set of downstream target genes important for overt rhythmicity (2, 14). One gene directly controlled by the WCC is os-4, which encodes the mitogen-activated protein kinase kinase kinase (MAPKKK) in the osmotically sensitive (OS) MAPK pathway (15). Rhythmic transcription of os-4 leads to rhythmic accumulation of the phosphorylated active form of the downstream p38-like mitogen-activated protein kinase (MAPK) OS-2 (16). In Sac...

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“…FRQ is produced, associates with the FRQ-interacting RNA helicase (FRH), and acts as the negative element of the TTFL shutting down its own expression by inhibiting the WCC (21). As FRQ is progressively phosphorylated, it decreases its negative effect on WCC, and it is finally degraded around the time that a new cycle of frq expression is initiated (23). Thus, this process can be visualized as daily oscillations in frq mRNA and protein levels.…”

mentioning

confidence: 99%

A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana

Hevia

Canessa

Müller-Esparza

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The circadian clock of the plant model Arabidopsis thaliana modulates defense mechanisms impacting plant–pathogen interactions. Nevertheless, the effect of clock regulation on pathogenic traits has not been explored in detail. Moreover, molecular description of clocks in pathogenic fungi—or fungi in general other than the model ascomycete Neurospora crassa—has been neglected, leaving this type of question largely unaddressed. We sought to characterize, therefore, the circadian system of the plant pathogen Botrytis cinerea to assess if such oscillatory machinery can modulate its virulence potential. Herein, we show the existence of a functional clock in B. cinerea, which shares similar components and circuitry with the Neurospora circadian system, although we found that its core negative clock element FREQUENCY (BcFRQ1) serves additional roles, suggesting extracircadian functions for this protein. We observe that the lesions produced by this necrotrophic fungus on Arabidopsis leaves are smaller when the interaction between these two organisms occurs at dawn. Remarkably, this effect does not depend solely on the plant clock, but instead largely relies on the pathogen circadian system. Genetic disruption of the B. cinerea oscillator by mutation, overexpression of BcFRQ1, or by suppression of its rhythmicity by constant light, abrogates circadian regulation of fungal virulence. By conducting experiments with out-of-phase light:dark cycles, we confirm that indeed, it is the fungal clock that plays the main role in defining the outcome of the Arabidopsis–Botrytis interaction, providing to our knowledge the first evidence of a microbial clock modulating pathogenic traits at specific times of the day.

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Decoupling circadian clock protein turnover from circadian period determination

Cited by 142 publications

References 54 publications

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa

Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana

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