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Two novel doubletime mutants alter circadian properties and eliminate the delay between RNA and protein in Drosophila.

ABSTRACT: Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. We have characterized two novel dbt mutants. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The PER profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for DBT during the PER monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription.

SUBMITTER: Suri V

PROVIDER: S-EPMC6772888 | BioStudies | 2000-01-01

REPOSITORIES: biostudies

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Project description:Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBT(K/R) with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBT(K/R) with the mutated NLS (DBT(K/R) NLS(-)) using a timGAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild-type flies. By contrast, expression of DBT(K/R) with the strong NLS (DBT(K/R) stNLS) using the timGAL4 driver lengthens period more strongly than DBT(K/R), with damped oscillations of PER phosphorylation and localization. Both DBT(K/R) and DBT(WT) without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBT(K/R) NLS(-) has lost its dominant negative property because it does not form normal clock protein complexes. DBT(WT) proteins with the same changes (NLS(-) and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBT(K/R) protein with both the stNLS and NLS(-) mutation does not affect circadian period, although it is nuclear, demonstrating that the lack of a dominant negative for the DBT(K/R) NLS(-) is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBT(K/R) but not of DBT(WT), suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.

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Two novel doubletime mutants alter circadian properties and eliminate the delay between RNA and protein in Drosophila.

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