Characterization of the novel sleep and circadian gene rogdi in drosophila melanogaster

Abstract

Sleep is a very dramatic and important phenomenon in human life. Most of mammal and non-mammal also need sufficient sleep in daily life. However, identification of sleep and wake mechanism is an unsolved problem. Mammal and insects share many character like decreased activity and response, increased sleep amount after disrupted sleep and prolonged inactivity. Fruit fly, Drosophila melanogaster, is a good model organism for clarifying sleep and wake mechanisms because this model system is easy to perform genetic experiment and huge size screening. In addition, many studies reveal that fruit fly has a similar molecular and neuronal sleep mechanism with mammalian organism. With Drosophila as a model, we identified a novel sleep and circadian regulating gene, the rogdi. We performed a forward genetic screening via NIG(National Institute of Genetics) P-element inserted library to find a novel sleep mutant. Through this screening, some short sleeping mutants were discovered and one of them had P-element near rogdi gene region. rogdi was reported previously as a learning and memory related gene. However, it was just mentioned briefly and wasn’t researched intensively. Hypomorphic rogdi mutant flies by P-element showed reduced sleep length especially in the nighttime. This mutant also had an arrhythmic circadian rhythm in DD condition, the short sleep phenotype is persisted in a period mutant background. An enhancer trap line in rogdi gene region showed its expression in sleep regulatory neurons in fly brains, like mushroom body, dopaminergic neurons and Pigment-dispersing factor (Pdf) expressing neurons. Furthermore, we overexpressed wild-type ROGDI via UAS-ROGDI with several Gal4 lines to narrow down sleep regulating neuron mediated by Rogdi. We found that expression of wild-type ROGDI in circadian neurons was not sufficient to recover their short sleep behavior in rogdi mutant background. Otherwise, overexpression in dopaminergic neuron by tyrosine hydroxylase (TH)-expressing Gal4 successfully rescued nighttime sleep in rogdi mutant. In addition, overexpression in octopaminergic neuron via tyrosine dehydroxylase2 (TDC2)-expressing Gal4 also restored sleep. TH is the enzyme responsible for converting L-tyrosine to L-3,4 dihydroxyphenylalanine ( L-DOPA). TH is known as rate-limiting enzyme for catalyzing dopamine (DA) from L-tyrosine. In rogdi mutant, TH expression level was elevated, especially in nighttime. Consequently, dopamine level is also increased. We also check mRNA level of several gene related with dopamine regulation and octopamine regulation. Interestingly, we found TH level and oamb mRNA level is increased in rogdi mutant. We clarified that ROGDI protein associates with RNA-binding proteins in a RNA-dependent manner. These data proposed that ROGDI is a novel regulator in dopamine signaling to promote sleep through TH translation interuption. Human has a rogdi homolog reported due to Kohlsch"utter-Tonz syndrome (KTS). KTS show mental retardation, epilepsy, amelogenesis imperfecta and developmental delay. Short sleep phenotype of rogdi mutant is restored via overexpression of human-ROGDI in adult brain. This result provides that fly can be a KTS model organism for studying.