Belle and Aurora A are novel components of the circadian machinery in Drosophila melanogaster
Year: 2011
Type: dissertation
Abstract: The circadian clock is a self-sustained system that synchronizes the physiology and behaviour of most organisms with the light-dark (LD) cycles. In humans, several pathological conditions, including cancer, are related to the deregulation of the circadian clock. Drosophila melanogaster is one of the most important models for the study of the circadian mechanism. The proteins Timeless (Tim) and Period (Per) are two main components of the master clock, which is located in the brain. The synchronization of the circadian clock to environmental changes is mostly achieved by blue light-dependent degradation of Tim, via the photoreceptor Cryptochrome (Cry). Cry mediates the daily resetting of the clock by light, binding to Tim and targeting it for degradation via proteasome. The oscillator is able to keep circadian time even in the absence of external stimuli and consists of a network of genes that function in transcriptional/translational feedback loops. Among the post-translational modification that regulate clock proteins amount, localization and activity, phosphorylation is likely the most important one. We have initiated a study in order to identify new molecular partners for Cry, both in light and dark. 1) The search for new partners of Cry by co-IP and mass spectrometry led to the identification of Belle, an ATP-dependent RNA helicase. Belle is expressed in the glial cells in fly adult brain, where also Cry is expressed. Moreover, the expression of this RNA-helicase oscillates in light-dark cycles (LD) and constant dark conditions (DD) in wild-type flies. Moreover, flies mutant for this gene do not show the typical rhythmic activity profile and show an impairment in Per expression in a subset of circadian neurons. Thereafter the downregulation of the gene causes reduced photophobicity in 3rd instar larvae and significant defects in the locomotor activity, suggesting an impairment of the circadian clock. Our data suggest that Belle is a novel “clock” protein involved in the circadian machinery of Drosophila, where it could act in the post-transcriptional control of circadian components. 2) It has been shown that the C-terminus of Cry contains putative targets for phosphorylation that play a role in the modulation of protein activity. Cry activation by light could be due to a conformational change or a release of a repressor. Phosphorylation could play an important role in the light dependent activation of the protein. In fact, AtCry1 and AtCry2 in plants (Arabidopsis thaliana) and mCry2 in mammals are phosphorylated after light exposure. in vitro experiments, performed in our lab, showed that Drosophila Cry is a putative target of the Serine-Threonine kinase Aurora A. Moreover, in silico analyses revealed that Cry C-terminus seems to contain consensus motifs which could be a target for this kinase. In particular, two Serine residues belonging to the SLIT and SNEE kinase consensus motifs in the C-terminal region result specifically phosphorylated. We have conducted a study in order to verify the in vivo role for Aurora A in Cry regulation. Analysis of single and double transgenic mutants for the residues target of phosphorylation seem to exclude a role for this sites in mediating Cry degradation after light exposure. However, experiments performed in flies over-expressing Aurora A show modifications in the circadian control of their rhythmic behaviour and display an increased degradation of Cry after light exposure. Taken together, these data suggest a putative implication of Aurora A in the regulation of Cry activity.
Author: Elena Carbognin
Language: English
Subfield: Endocrine and Autonomic Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W160609392