Dataset Information

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-literature | 2000 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Suri V V Hall J C JC Rosbash M M

The Journal of neuroscience : the official journal of the Society for Neuroscience 20001001 20

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, co ...[more]

PMID: 11027213

Similar Datasets

Project description:While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

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.

Project description:Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

Project description:Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, ?) is influenced by temperature in some of these mutants, whereas ? is temperature-independent in other mutant lines as in wild-type flies. This, so-called "temperature compensation" ability is compromised in the mutant timeless allele "ritsu" (tim rit ), and, as we show here, also in the tim blind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of ? and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer ? at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets