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Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, we constructed the first circadian “translatome” for astrocytes. This analysis identified 293 “cycling genes” in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, we show that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns.

The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation., Author Summary The CKI family of serine/threonine kinase regulates diverse cellular processes, through binding to and phosphorylation of a variety of protein substrates. In mammals, mutations in two members of the family, CKIε and CKIδ were found to affect circadian period length, causing phenotypes such as altered circadian period in rodents and the Familial Advanced Sleep Phase Syndrome (FASPS) in human. The Drosophila CKI δ/ε homolog DOUBLETIME (DBT) is known to have important roles in development and circadian clock function. Despite extensive studies of DBT function, little is known about how its expression is regulated. In a previous genome-wide study, we identified dbt mRNAs as potential targets of the LARK RBP. Here we describe a detailed study of the regulation of DBT expression by LARK. We found that LARK binds to and regulates translation of dbt mRNA, promoting expression of a smaller isoform; we suggest this regulatory mechanism contributes to circadian period determination. In addition, we have identified a dbt mRNA that exhibits light-induced changes in translational status, in a LARK-dependent manner. Our study is the first to analyze the translational regulation of DBT, setting the stage for similar studies in other contexts and model systems.

Despite substantial progress in defining central components of the circadian pacemaker, the output pathways coupling the clock to rhythmic physiological events remain elusive. We previously showed that LARK is a Drosophila RNA-binding protein which functions downstream of the clock to mediate behavioral outputs. To better understand the roles of LARK in the circadian system, we sought to identify RNA molecules associated with it, in vivo, using a three-part strategy to (1) capture RNA ligands by immunoprecipitation, (2) visualize the captured RNAs using whole-genome microarrays, and (3) identify functionally relevant targets through genetic screens. We found that LARK is associated with a large number of RNAs, in vivo, consistent with its broad expression pattern. Overexpression of LARK increases protein abundance for certain targets without affecting RNA level, suggesting a translational regulatory role for the RNA-binding protein. Phenotypic screens of target-gene mutants have identified several with rhythm-specific circadian defects, indicative of effects on clock output pathways. In particular, a hypomorphic mutation in the E74 gene, E74(BG01805), was found to confer an early-eclosion phenotype reminiscent of that displayed by a mutant with decreased LARK gene dosage. Molecular analyses demonstrate that E74A protein shows diurnal changes in abundance, similar to LARK. In addition, the E74(BG01805) allele enhances the lethal phenotype associated with a lark null mutation, whereas overexpression of LARK suppresses the early eclosion phenotype of E74(BG01805), consistent with the idea that E74 is a target, in vivo. Our results suggest a model wherein LARK mediates the transfer of temporal information from the molecular oscillator to different output pathways by interacting with distinct RNA targets.

Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsα, or the Gardos K+ channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K+ channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 μg of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera., Synopsis Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods, and insects have previously been implicated as vectors of this disease. Here researchers report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease. Furthermore, although ingestion of cholera toxin results in massive diarrhea in mammals, these researchers have found that ingestion of purified cholera toxin is not lethal to the fly. However, when co-ingested with a pathogenic strain of V. cholerae carrying a deletion of the cholera toxin genes, cholera toxin is lethal. These findings not only demonstrate the utility of D. melanogaster as an accurate, inexpensive model for elucidation of the host-pathogen interaction and identification of inhibitors of the action of cholera toxin; they also suggest that V. cholerae carries additional virulence factors that enable intoxication of an arthropod host. Based on these findings, the researchers suggest that the fly or a related arthropod may be a true host of V. cholerae in nature.