PhD Scientific Days 2022

Budapest, 6-7 July 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

Role of RasGEF-mediated pathway in circadian rhythm regulation

Orsolya Sárkány, Department of Physiology, Budapest
Anita Szőke, Department of Physiology, Budapest
Dr. Norbert Gyöngyösi, Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Budapest
Dr. Káldi Krisztina, Department of Physiology, Budapest

Text of the abstract

Introduction
The circadian rhythm is a crucial mechanism of biological adaptation to daily changes in the environment. The endogenous rhythm is generated by molecular oscillators. Environmental factors can entrain the rhythm, one of them is nutrient availability. Through the so-called metabolic compensation, the oscillator works at a nearly constant rate at different substrate levels, providing a period length of ~24h. The importance of the relationship between metabolism and circadian clock is supported by a large set of clinical and experimental data.
Aims
RAS-mediated cellular pathways are involved in responses to changes in nutrient supply in several organisms including mammals. Our team identified RAS2 as an important component of metabolic compensation of the clock of Neurospora crassa. Our current work addresses the role of a RasGEF protein in the interaction between circadian rhythm and nutrient levels.
Methods
Both circadian rhythm and cellular control of metabolism are well-conserved mechanisms. Therefore, we used in our experiments the circadian model organism, the Neurospora crassa. Phase and period of the circadian rhythm were determined by analysis of the conidiation rhythm in race tubes. A rescue strain was generated by transforming the rasgef-ko strain with a construct coding for a Flag-tagged version of RasGEF. Expression of clock components and rasgef at the RNA and the protein levels was determined by quantitative real-time PCR and Western blot, respectively. Time-dependent regulation of the rasgef promoter was followed by in vivo luciferase assay.
Result and conclusion
The phase of conidiation was delayed in both light-dark and temperature cycles, and under constant conditions the period was longer in the rasgef mutant compared to the wild type strain. At the molecular level, expression of the positive clock components was increased and less sensitive to changes of the glucose concentration. Luciferase expression controlled by the rasgef promoter was rhythmic, suggesting that rasgef is a clock-controlled gene (ccg). rasgef RNA levels were glucose repressible, also indicating the role of RasGEF in the adaptation of the clock function to nutrient availability.
We suggest that rasgef is a novel glucose repressible ccg which plays a role in the metabolic regulation of the circadian clock of Neurospora crassa.
Funding
SE250+ and FE0301ELET TKP-EGA-25