The overall goal of this proposal is to understand how a circadian pacemaker accommodates new phase alignments in response to seasonal changes in ambient temperature and day length (photoperiod). A major foundation for the proposed specific aims is based on our prior work showing that the thermosensitive and photosensitive splicing of the 3'-terminal intron from the D. melanogaster period (per) RNA (termed dmpi8; D. melanogaster dper intron 8) acts as a multi-modal """"""""seasonal sensor"""""""" conveying calendar information. High splicing efficiency of dmpi8 on seasonally cold days (i.e., low temperatures and short photoperiods) advances the timing of 'evening' activity, endowing flies with the ability to manifest daytime activity despite the early onset of dusk. Conversely, low splicing efficiency of dmpi8 on warm days ensures that flies minimize activity during the hot midday sun and are mainly nocturnal, lowering the risks associated with desiccation. We also showed that the norpA (no receptor potential A) gene encoding phospholipase C (PLCbeta), modulates the temperature and clock control of dmpi8 splicing efficiency. These findings identified a novel non-photic role for norpA. In this application we propose to determine how temperature, light, clock factors and NORPA co-regulate the splicing efficiency of dmpi8, and the contribution of this molecular event to seasonal adaptation in rhythmic behavior. Major objectives are to identify cis-acting elements that mediate thermosensitivity of dmpi8 splicing and investigate the role of the PLC/phosphatidylinositol signaling cascade. Moreover, we will analyze the clockworks and behavioral rhythms in natural populations of several different Drosophila species isolated from a wide range of latitudes. Preliminary findings suggest that per 3'-terminal introns are targets for natural selection in the adaptation of circadian clocks to geographical variations in seasonal climates. To accomplish these goals we propose to take multifacted experimental strategies including biochemical, tissue culture and whole animal approaches. Our studies will add insights into seasonal adaptation of clock function and reveal how norpA participates in calibrating molecular and behavioral thermoresponses in D. melanogaster.
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