PhD Scientific Days 2024

Molecular Medicine I.

Role of a RASGEF-mediated pathway in the metabolic compensation of the circadian clock

Text of the abstract

Introduction: The circadian rhythm regulates physiological functions over a roughly 24-hour period. This mechanism ensures adaptation to the changing environment and is based on the conserved function of a transcription-translation feedback loop. Metabolic compensation of the rhythm allows the endogenous oscillator to work with similar periods at different nutrient levels.
The RAS pathway is involved in the regulation of cellular processes such as proliferation, as well as nutrient sensing. Our previous results indicated the role of the RAS2 in the metabolic compensation of the circadian clock in the model organism Neurospora crassa.
Aim: Examination of the role of a putative RASGEF in the regulation of the circadian clock and its adaptation to low nutrient conditions.
Method: As a circadian output, the conidiation rhythm of Neurospora was monitored via race-tube assay to examine the phase and period of the clock. A rescue strain was generated by transforming the rasgef-ko strain with a FLAG-tagged version of RASGEF. RNA and protein levels were analyzed using qRT-PCR and Western blot, respectively. A rasgef-luc strain expressing luciferase under the control of the putative rasgef promoter was generated. Human U2OS cells expressing luciferase under the control of the promoter of Bmal1 (positive factor of the circadian clock) were cultured under different glucose conditions and treated with inhibitors of the mammalian homolog of the RASGEF-mediated pathway.
Results: The rasgef-ko Neurospora strain displayed a longer conidiation rhythm than the control and showed disturbed metabolic compensation. rasgef is a clock-controlled gene encoding a protein that shows glucose-dependent phosphorylation and can be detected in both the cytosol and the nucleus. In rasgef-ko, expression of the positive clock components exhibits reduced sensitivity to glucose deprivation compared to the wt. In U2OS-Bmal1-luc cells, phase and period remain stable even under low glucose conditions. Inhibition of SOS1 and ERK highlights the role of SOS-1/ERK-mediated signaling in the maintenance of clock function under low glucose conditions.
Conclusion: We suggest that rasgef modulates the circadian clock in a glucose-dependent manner, thereby supporting robust circadian function in both fungi and mammals, particularly under low glucose conditions.
Funding: SE250+, OTKA K132393, TKP2021-EGA-25