Characterization of a novel circadian rhythm gene, twenty-four, in drosophila

Abstract

The earth’s rotation results in daily environmental changes, such as light/dark and temperature. To deal with these changes better, a variety of organisms have evolved circadian clock system. The circadian rhythms are generated by cell-autonomous and endogenous clock system. The rhythm persists without time cues and can be tuned by the external time signal. Using UAS/GAL4 systems, we performed a genetic screen for circadian locomotor activities. Through this screen, we identified several candidates that showed long periods of behavior. In this study, we report on the analysis of one of the candidates, CG4857 that we named $\It{twenty-four}$ ($\It{twf}$, 4+8+5+7=24). Targeted expression of $\It{twf}$ by circadian specific GAL4 drivers resulted in a long period. Two strong hypomorphic $\It{twf}$ alleles as well as RNAi-mediated $\It{twf}$ knockdown in clock cells showed in weak but long periods. The complementation test between twf hypomorphs and deficiencies confirmed that mutations of $\It{twf}$ responsible for the circadian phenotypes. To reveal the underlying molecular mechanism, we examined the expression of circadian genes in the brain. As a result, dramatic reduction of PERIOD protein levels was observed in $\It{twf}$ hypomorphs. Restoring TWF expression in the ventral lateral neurons is sufficient to rescue behavioral rhythms and PER cycling. $\It{per}$ transgenes lacking the per promoter or lacking a domain important for PER degradation were influenced by $\It{twf}$. These results suggest the post-transcriptional function of $\It{twf}$. TWF was associated with per and tim transcripts and the poly(A)-binding protein (PABP), respectively. Furthermore, TWF was sedimented with polyribosomes in sucrose gradient. Using RNA tethering assay in S2 cells, we found that TWF activates reporter expression, which fused with $\It{per}$ 3’UTR. These findings suggest that twf is required for circadian locomotor activities and specifically activates PER expression in pace...