• Search for new genes involved in the circadian clock of Drosophila melanogaster: molecular and behavioural analysis of putative - CRYPTOCHROME

Abstract: In Drosophila melanogaster CRYPTOCHROME (dCRY) is a blue light photoreceptor involved in the photic input pathway to the circadian clock. It mediates the daily resetting of the brain clock by light, a fundamental step in the generation of circadian rhythmicity. In fact, upon light stimuli, dCRY specifically binds to TIMELESS  (dTIM), a cardinal component of the clock machinery, targeting it to degradation via proteasome. We previously hypothesized that the activation of dCRY by light is mediated by specific “regulators” that bind the C-terminus of the protein, which has been proven to regulate the light-dependence of dCRY activity. This hypothesis was supported by the observation that the C-terminus of dCRY is an hotspot for molecular interactions. In fact, by in silico analysis and experimental validation we could identify, in this small region, several linear motifs involved in protein-protein interaction, through domains like PDZ and TRAF2 present in proteins that participate in several processes, as DNA binding, signal transduction, protein-protein interaction and targeting proteins for proteasome-mediated degradation. It has been also observed that in the peripheral clocks dCRY has a fundamental role in the circadian pacemaker, where it acts as transcriptional repressor. This activity is not light dependent, as it is required for normal rhythmicity in free-running conditions. It can be then hypotesised that different regulatory elements could modulate dCRY activity in peripheral and brain oscillator cells.

We undertook the research of putative partners of dCRY by two different strategies: (a) the screening a Drosophila adult head cDNA library by yeast-two-hybrid assay and (b) the co-immunoprecipitation experiments on transgenic flies overexpressing dCRY and dissection of the complexes by mass  spectrometry analysis. In this way we have identified proteins that could be either the regulators of dCRY light-dependent activity or involved in other activities of dCRY, independent from light. This project aims to analyse in details some of the interactions observed. In our experiments dCRY has been found to interact with proteins involved in the light transduction pathway in Drosophila, like Rh1-NINAE (neither inactivation nor afterpotential E, the main photopigment in the compound eye), NINAC (neither inactivation nor afterpotential C, a motor activity protein similar to miosin, with kinase activity) and RdgA (retinal degeneration A, a diacylglycerol kinase, involved in Protein Kinase C-PKC activation). dCRY is highly expressed in Drosophila eyes, but a role for this receptor in the visual system has never been proven. Among the putative partners of dCRY we found also a protein containing JAB_MPN and Mov34 domains, typical of proteasomal subunits, probably involved in the regulation of dCRY stability. Moreover dCRY has been found to interact also with hRNP, a heterogeneous nuclear ribonucleoprotein involved in mRNA maturation. This data supports the already observed function of dCRY as transcriptional repressor in the peripheral clocks. Whereas the involvement of dCRY in the circadian photoreception is extensively studied, little is know about its role as part of the main feedback loop. We therefore aim to understand if this regulative activity of dCRY is specifically achieved by regulation of transcription, as previously hypothesized, or it might be explicated also via a post-transcriptional control mechanism.  In this project we plan to characterise in details these interactions, that is: 1) confirm them by co-immunoprecipitation and western blot assays; 2) analyse the stability of this complexes during the circadian cycle; 3) co-localise dCRY and its partners in the Drosophila head (both central and peripheral clocks); 4) analyse the expression and the stability of dCRY in transgenic flies where the corresponding genes have been silenced by using a RNAi approach; 6) analyse the circadian behaviour of the RNAi flies.

Scientific Coordinator