InteractiveFly: GeneBrief

jetlag: Biological Overview | References

Organisms ranging from bacteria to humans synchronize their internal clocks to daily cycles of light and dark. Photic entrainment of the Drosophila clock is mediated by proteasomal degradation of the clock protein Timeless. Mutations have been indentified in jetlag (a gene coding for an F-box protein with leucine-rich repeats) that result in reduced light sensitivity of the circadian clock. Mutant flies show rhythmic behavior in constant light, reduced phase shifts in response to light pulses, and reduced light-dependent degradation of Tim. Expression of Jet along with the circadian photoreceptor Cryptochrome (Cry) in cultured S2R cells confers light-dependent degradation onto Tim, thereby reconstituting the acute positive response of the circadian clock to light in a cell culture system. These results suggest that Jet is essential for resetting the clock by transmitting light signals from Cry to Tim (Koh, 2006).

In the course of characterizing rest:activity rhythms of various fly strains, a strain was discovered with anomalous activity patterns in constant light (LL). Whereas wild-type flies became arrhythmic after a day or two in LL, the mutant flies were rhythmic for more than a week. Although the mutants could be entrained to light:dark (LD) cycles, they took longer to be re-entrained to a new schedule than wild-type flies, and so the mutation was named jetlag. The behavior of jet flies in LD and in constant darkness (DD) conditions was normal. These phenotypes are reminiscent of those of cry mutants and suggest a defect in circadian photoreception (Koh, 2006).

Using meiotic recombination and deficiency mapping strategies, the mutation was mapped to a small region containing 18 genes on the left arm of the second chromosome. One of these genes, CG8873, encodes an F-box protein with leucine-rich repeats (LRRs), a putative component of a Skp1/Cullin/F-box (SCF) E3 ubiquitin ligase complex. The coding region of the gene was sequenced in 13 strains, including some wild-type strains, the original mutant strain, and several other strains that did not complement the original mutation for the LL phenotype. In six of the seven mutant strains, a phenylalanine-to-isoleucine substitution was found in a conserved LRR domain. In the remaining mutant strain, there was a serine-to-leucine substitution in an adjacent LRR domain. The two mutations will be referred to as common and rare (c and r), respectively. The two alleles did not complement each other, nor did they complement chromosomal deletions that remove the jet locus (Koh, 2006).

The Jet protein contains an N-terminal F-box domain thought to be involved in binding the Skp1 component of the SCF complex, as well as seven LRRs constituting a protein-protein interaction domain thought to be involved in target recognition. Functions of the mammalian F-box proteins with highest similarity to Jet (F-box and LRR protein 15) have yet to be determined (Koh, 2006).

Almost all (>96%) of the jet^r and jet^c flies had rhythmic behavior in LL, whereas very few of the wild-type control flies did. In contrast, the mutants' behavior was indistinguishable from wild-type behavior in DD, which suggests that the mutants have a largely intact circadian system with a specific defect in the light input pathway. Consistent with its limited role in free-running rhythms, the jet mRNA does not cycle in a circadian fashion. The reduced light sensitivity of jet mutants is similar to that of cry mutants; however, unlike cry mutants, jet mutants showed rhythmic activity of a luciferase reporter for a clock gene, period (per), in DD, which suggests that their peripheral clocks function normally. Because luciferase is assayed in whole flies and therefore reports the activity of multiple peripheral clocks, rhythmic luciferase activity in jet mutants also indicates synchrony among these clocks. Peripheral clocks can be entrained to an LD cycle via Cry-independent pathways, which may account for the synchrony of peripheral clocks in jet mutants. Loss of per-luciferase cycling in cry mutants most likely occurs because Cry, in addition to its role as a circadian photoreceptor, has a role in the regulation of core clock components in the periphery (Koh, 2006).

To characterize the behavioral light sensitivity of jet mutants in more detail, phase shifts were measured in response to brief light pulses at night. jet mutants had significantly reduced phase shifts relative to wild-type control flies. Expression of wild-type Jet from a UAS-jet transgene under the control of a cry- or tim-Gal4 driver partially rescued the mutant phenotype. The increase in phase shifts was greater with tim-Gal4 than with cry-Gal4, probably because the former is a stronger driver. Together with the sequence data described above, the rescue results provide strong evidence that the mutations in the jet locus are responsible for the observed mutant phenotypes (Koh, 2006).

To determine the molecular correlates of the behavioral defects, the changes in TIM levels were examined in central clock neurons after brief light pulses. Light-dependent degradation of Tim was substantially reduced in jet mutants and was restored in rescued flies expressing the UAS-jet transgene, which suggests that the behavioral defects in the mutants are mediated by defects in Tim degradation. Light-dependent Tim degradation was also reduced in head extracts of mutants (fig. S2), implying that Jet facilitates Tim degradation in the peripheral clock in the eye as well (Koh, 2006).

To further explore the role of Jet in Tim degradation, an S2R+ Drosophila embryonic cell line was used. Unlike in the fly, in S2R+ cells, Tim does not degrade in response to light. To test whether Jet is the crucial component missing in these cells, Jet was expressed with the use of a constitutive promoter. The Jet protein had little effect on Tim levels in the dark, but it rapidly reduced Tim levels upon light exposure. This light-induced degradation of Tim required coexpression of Cry and was blocked by a proteasome inhibitor, MG132. Jet did not promote degradation of another core clock protein, PER, demonstrating specificity for its target selection. In addition, light-dependent degradation of Cry did not require Jet, although it was facilitated by Jet in the presence of Tim. The Jet protein itself was also not affected by light in S2R+ cells (Koh, 2006).

One of the mutant versions of the protein (the r allele) was significantly less effective than wild-type Jet at promoting Tim degradation. The r mutation reduced the stability of the Jet protein, which may explain its mutant phenotype. The other mutant allele (c) was also less effective than the wild-type allele, although the difference was not statistically significant. The r mutation is in a residue conserved among insects and mammals, but the c mutation is in a residue conserved only among insects, which may explain the stronger effects of the r allele in both behavioral and molecular assays (Koh, 2006).

Wild-type Jet protein also promoted ubiquitination of Tim in cultured cells. In the presence of wild-type Jet and Cry, a significant increase in Tim ubiquitination was observed after only 10 min of exposure to light. Mutant proteins, especially the r allele, were less effective at ubiquitination of Tim. Consistent with its role as a component of an SCF complex, Jet interacts with SkpA, one of several Skp1 homologs in Drosophila. In addition, Jet physically associates with Tim, and the association is stronger in light than in dark (Koh, 2006).

Flies share many of the core clock components with mammals, but their mechanism for light-induced phase resetting appears to differ. Circadian photoreception in mammals relies on adenosine 3',5'-monophosphate response element–binding protein (CREB)–mediated induction of mPer-1 transcription and does not appear to involve Cry. Fly circadian photoreception resembles that of plants, where Cry functions as a circadian photoreceptor, although the mechanism is somewhat different from that of Drosophila Cry. Notably, the plant F-box protein ZEITLUPE mediates dark-dependent degradation of the clock protein TimING OF CAB EXPRESSION 1 (Koh, 2006).

jet mutants did not show any detectable defects in the free-running rhythm in constant darkness. It is proposed that the Drosophila circadian system uses two separate mechanisms for controlling Tim levels: a clock-controlled one for maintaining rhythm in the dark, and a light-dependent one for entraining the clock to the photic environment. Both mechanisms use SCF complexes but with distinct F-box proteins: SLIMB for the clock-controlled mechanism and Jet for the light-dependent mechanism (Koh, 2006).

This study has identified a component of the Drosophila light entrainment pathway that is critical for light-induced degradation of Tim. Single amino acid substitutions in Jet lead to molecular and behavioral defects in light entrainment. These results suggest the following model of how light resets the clock in Drosophila. Upon light exposure, Cry undergoes conformational change, allowing it to bind Tim. Tim is then modified by phosphorylation, which allows Jet to target Tim for ubiquitination and rapid degradation by the proteasome pathway (Koh, 2006).

Search PubMed for articles about Drosophila Jetlag

Koh, K., Zheng, X. and Sehgal, A. (2006). JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312(5781): 1809-12. 16794082

date revised: 6 October 2007

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