Identifying the function of nonsense-mediated mRNA decay in regulation of circadian rhythm in Drosophila melanogaster

Abstract

Most living organisms have innate biological clock that has evolved to follow the 24-hr rhythm made by the earth’s rotation. Drosophila melanogaster has molecular feedback loops as well as similar network of hormonal and nervous systems commonly shared with mammals. This makes it an excellent model organism for studies in circadian clock and sleep regulation. In this research, we have characterized another circadian rhythm controlling mechanism conserved in both flies and mammals. While screening for post-transcription regulatory genes that influence Drosophila’s rhythmic activity, we discovered that SMG5 and SMG6 play an important role in circadian regulation without affecting the development of neurons. We confirmed that it is their function in nonsense-mediated mRNA decay that regulates flies’ activity. Nonsense-mediated mRNA decay (NMD) is one of the major mRNA surveillance pathway orchestrated by a number of components, recruited by premature termination codon. Fruit flies lose their rhythmicity when one or more of these components are in insufficient amount or excessively produced. However, neither the expression level nor the oscillation of the key circadian genes was different from those of the flies with normal behavior. This implies that the altered rhythmicity triggered by impaired NMD has no association with the core clock system. Instead, this mRNA degradation pathway affected the level of dCREB2, more specifically of dCREB2-b, and influence flies’ rhythm via output pathway. Expression of normal dCREB2-b restored mutant flies’ rhythmicity.

Nonsense-mediated decay is also responsible for quality control of gene expression under stress conditions in both organismal and cellular level. We have carried out RNA sequencing of the brains of flies sleep-deprived for different lengths of time, and analyzed for most affected functions. We continued to look at the homeostatic sleep phenotype of the flies with defects in genes that were validated to have excessive level change induced by sleep deprivation.