Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3 splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.
Circadian fluctuations in per mRNA and protein are central to the operation of a negative feedback loop that is necessary for setting the free-running period and for entraining the circadian oscillator to light-dark cycles. In this study, per mRNA cycling and locomotor activity rhythms were measured under different light and dark cycling regimes to determine how photoperiods affect the molecular feedback loop and circadian behavior, respectively. These experiments reveal that per mRNA peaks in abundance 4 h after lights-off in photoperiods of <16 h, that phase shifts in per mRNA cycling and behavioral rhythmicity occur rapidly after flies are transferred from one photoperiod to another, and that photoperiods longer than 20 h abolish locomotor activity rhythms and leave per mRNA at a median constitutive level. These results indicate that the per feedback loop uses lights-off as a phase reference point and suggest (along with previous findings for per 01 and tim 01 ) that per mRNA cycling is not regulated via simple negative feedback from the per protein.Circadian rhythms in biochemical, physiological, and behavioral phenomena are a fundamental adaptation of both prokaryotic and eukaryotic organisms to environmental changes that occur over a 24-h period. These rhythms are driven by an endogenous clock that continues to operate under constant environmental conditions. The timekeeping component of the clock, or pacemaker, maintains a periodicity that can be hours longer or shorter than 24 h, and it is synchronized to local time by such environmental signals as light and dark. A stable phase relationship between the pacemaker and its Zeitgeber is clearly a prerequisite if preprogrammed biological changes are to be appropriately timed to daily environmental changes. Physiological and behavioral experiments have been used to determine how the clock adjusts its phase to circadian cycles composed of different proportions of light and dark. During these different photoperiodic conditions, the phase of the circadian pacemaker (and its physiological and behavioral output) is altered so that a stable phase relationship is maintained (26). Because of the lack of measurable pacemaker components, however, these physiological studies could not address the molecular mechanism by which the clock adjusts its phase to accommodate different environmental light-dark (LD) cycles.Genetic screens for rhythm mutants have been used to identify components of the circadian pacemaker. Mutations in the per gene from Drosophila melanogaster can shorten (per S and per) circadian rhythms of locomotor activity and eclosion during constant dark (DD) conditions (16,17) and alter the phase of locomotor activity and eclosion rhythms during LD cycling conditions (3,10,11,17,30). These behavioral effects of the per mutants are paralleled at the molecular level by circadian fluctuations in the abundance of per mRNA and per protein (PER) (12, 41). These fluctuations in per mRNA and protein levels compose a negative feedback loop in which per mRNA serves ...
In Drosophila melanogaster, the emergence of adults from their pupal cases (eclosion) is gated by the circadian clock such that it occurs during a window of approximately 8-10 h starting 1-2 h before lights-on in 12-h light:12-h dark cycles (LD). This gate is shifted several hours earlier by the clock mutant per(s), indicating that the clock controls the phase of eclosion under these conditions. Both the day and the time of eclosion are determined by the interplay between developmental state and the circadian clock. At a certain phase of the circadian cycle, the circadian clock, either directly or through some circadian clock-controlled mechanism, measures development state, and those pharate adults that have reached a certain developmental state by this phase eclose during the first available gate, while those that have not wait until a subsequent gate. Using wing pigmentation as a late developmental state marker, an early boundary for when the circadian clock assesses developmental state occurs roughly at the time when lights go out during LD cycles. This event is shifted several hours earlier in per(s), showing that it is under circadian control. A fly's developmental state at the time of developmental assessment also influences when eclosion will occur (during the gate) in that flies whose wings have become pigmented early (12-24 h before assessment) will eclose earlier in the gate than those whose wings become pigmented late (0-12 h before assessment). These data suggest that the circadian clock (or some clock-controlled mechanism) measures developmental state (wing pigmentation) in wild-type flies between lights-off and expression of the first clock-regulated marker approximately 4-5 h before eclosion and that the developmental state of the fly determines both which gate is chosen for eclosion and when eclosion occurs during that gate.
The period ( per) gene is an essential component of the circadian timekeeping mechanism in Drosophila. This gene is expressed in a circadian manner, giving rise to a protein that feeds-back to regulate its own transcription. A 69 bp clock regulatory sequence (CRS) has been identified previously upstream of the period gene. The CRS confers wild-type mRNA cycling when used to drive a lacZ reporter gene in transgenic flies. To determine whether the CRS also mediates proper developmental and spatial expression and behavioral rescue, we used the CRS to drive either lacZ or per in transgenic flies. The results show that the CRS is able to activate expression in pacemaker neuron precursors in larvae and essentially all tissues that normally express per in pupae and adults. The CRS is sufficient to rescue circadian feedback loop function and behavioral rhythms in per 01 flies. However, the period of locomotor activity rhythms shortens if a stronger basal promoter is used. This study shows that regulatory elements sufficient for clock-dependent and tissue-specific per expression in larvae, pupae, and adults are present in the CRS and that the period of adult locomotor activity rhythms is dependent, in part, on the overall level of per transcripts. Key words: Drosophila; circadian clock; transcriptional regulation; behavior; period gene; developmental expressionIn Drosophila melanogaster, an autoregulatory feedback loop in gene expression is a central feature of the circadian timekeeping mechanism. In this feedback loop, the period ( per) and timeless (tim) genes are rhythmically expressed such that circadian fluctuations in per and tim mRNA levels are controlled by fluctuating levels of PER and TIM proteins (Rosato et al., 1997;Hardin and Sehgal, 1998). As PER and TIM accumulate, they bind to each other and move into the nucleus (Vosshall et al
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