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

Abstract

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.

Adaptation to starvation requires a flexible circadian clockwork inNeurospora crassa

“…Hence, we performed subcellular fractionation on our LL samples (Figure 1F). In accordance with previous data (Cheng et al, 2005; Gyongyosi et al, 2017; Schafmeier et al, 2006), the majority of FRQ was in the cytosol fraction, and its distribution did not change markedly upon glucose-deprivation. In contrast, WC proteins were virtually absent from the cytosol of starved cells, whereas their nuclear concentrations were similar to those in the control cells.…”

“…Nutrient compensation of the circadian rhythm and thus timely regulation of physiology could therefore be a prerequisite for appropriate adaptation. Short glucose deprivation does not affect expression of the core clock proteins and this maintenance of the protein levels was considered essential for nutrient-compensation (Emerson et al, 2015; Gyongyosi et al, 2017; Sancar et al, 2012). Our experiments show that the circadian TTFL can function with substantially altered levels and stoichiometry of its key elements.…”

“…Extraction of Neurospora protein, Western blots (Gyongyosi et al, 2013; Schafmeier et al, 2006) and subcellular fractionation (Gyongyosi et al, 2017; Luo et al, 1998) was performed as described earlier. In subcellular fractionation same amount of cytosolic and nuclear fractions were analyzed.…”

“…The WCC also acts as the major photoreceptor of Neurospora and transduces light information to the clock (Froehlich et al, 2002; He et al, 2002). In Neurospora , short-term (0-16 hr) glucose deprivation triggers compensatory mechanisms at the transcriptional and posttranscriptional levels that maintain expression levels of the core clock proteins, thereby keeping period length constant (Adhvaryu et al, 2016; Emerson et al, 2015; Gyongyosi et al, 2017; Olivares-Yanez et al, 2016).…”

A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

“…Over-compensation was also found in loss-of-function mutants of an RNA helicase, PRD-1, which normally localizes to the nucleus only under high glucose conditions (Emerson et al, 2015). Nutrient sensing and signaling pathways should presumably also play a role in Nutritional Compensation of the clock, and RAS2 and cAMP signaling have been implicated (Gyöngyösi et al, 2017). Taken together, the current incomplete model for Nutritional Compensation in Neurospora assembled from random hits implicates transcriptional and post- transcriptional regulation of core clock factors by transcription factors, an RNA helicase, and cAMP signaling.…”