Mary A. Roberts

Cellular and molecular mechanisms of circadian control in insects

Abstract Genetic analysis in Drosophila melanogaster has identified molecules important for the function of insect circadian clocks, and this has resulted in the elaboration of explicit biochemical models of the clock mechanism. Comparable molecular genetic analysis coupled with neuroanatomical approaches has also delineated cellular elements of the circadian pacemaker controlling insect activity rhythms. However, not much is known about the transfer of temporal information from clock cells in the insect brain to downstream neural elements or other target cells that are regulated by the clock (i.e. clock output pathways). In this review, we focus on the insect literature, with special reference to the fruitfly D. melanogaster and the hawkmoth Manduca sexta, to discuss the candidate molecules, biochemical mechanisms and cell types implicated in the clock control of behavior.

The LARK RNA-Binding Protein Selectively Regulates the Circadian Eclosion Rhythm by Controlling E74 Protein Expression

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, E74BG01805, 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 E74BG01805 allele enhances the lethal phenotype associated with a lark null mutation, whereas overexpression of LARK suppresses the early eclosion phenotype of E74BG01805, 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.

Ribosomal S6 Kinase Cooperates with Casein Kinase 2 to Modulate the Drosophila Circadian Molecular Oscillator

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the casein kinase 2 regulatory subunit (CK2β), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism.

Molecular analysis of the Drosophila miniature-dusky (m-dy) gene complex: m-dy mRNAs encode transmembrane proteins with similarity to C. elegans cuticulin

Abstract. Mutations in the Drosophila miniature-dusky (m-dy) gene complex were first reported by Morgan and Bridges about 90 years ago. m-dy mutants have abnormally small wings, a phenotype attributed to a cell-autonomous reduction in the size of the epidermal cells comprising the differentiated wing. Using a molecular genetic approach, we have characterized the m-dy chromosomal interval and identified a pair of adjacent transcription units corresponding to m and dy. A dy mutant known as dyAnd has a single base substitution within the protein-coding region that is predicted to result in an amber stop codon and premature translational termination. We show that dy mRNA is expressed at two discrete periods during the life cycle – one during embryonic development and early larval instars, the second during adult development, coincident with wing differentiation. In agreement with the phenotypic similarity of m and dy mutants, sequence comparisons reveal a similarity between the predicted MINIATURE and DUSKY proteins, and indicate that the m and dy genes are members of a larger Drosophila gene family. Both m and dy, as well as other members of this superfamily, are predicted to encode transmembrane proteins with similarity to C. elegans cuticle proteins known as cuticulins. We postulate that m, dy and other members of this protein superfamily function as structural components of the Drosophila cuticulin layer. Such a role for m and dy products in wing differentiation is sufficient to explain the morphological phenotypes associated with m-dy mutants.

Cellular Requirements for LARK in the Drosophila Circadian System

RNA-binding proteins mediate posttranscriptional functions in the circadian systems of multiple species. A conserved RNA recognition motif (RRM) protein encoded by the lark gene is postulated to serve circadian output and molecular oscillator functions in Drosophila and mammals, respectively. In no species, however, has LARK been eliminated, in vivo, to determine the consequences for circadian timing. The present study utilized RNA interference (RNAi) techniques in Drosophila to decrease LARK levels in clock neurons and other cell types in order to evaluate the circadian functions of the protein. Knockdown of LARK in timeless (TIM)– or pigment dispersing factor (PDF)–containing clock cells caused a significant number of flies to exhibit arrhythmic locomotor activity, demonstrating a requirement for the protein in pacemaker cells. There was no obvious effect on PER protein cycling in lark interference (RNAi) flies, but a knockdown within the PDF neurons was associated with increased PDF immunoreactivity at the dorsal termini of the small ventral lateral neuronal (s-LNv) projections, suggesting an effect on neuropeptide release. The expression of lark RNAi in multiple neurosecretory cell populations demonstrated that LARK is required within pacemaker and nonpacemaker cells for the manifestation of normal locomotor activity rhythms. Interestingly, decreased LARK function in the prothoracic gland (PG), a peripheral organ containing a clock required for the circadian control of eclosion, was associated with weak population eclosion rhythms or arrhythmicity.

Genetic and biochemical strategies for identifying Drosophila genes that function in circadian control.

Explicit biochemical models have been elaborated for the circadian oscillators of cyanobacterial, fungal, insect, and mammalian species. In contrast, much remains to be learned about how such circadian oscillators regulate rhythmic physiological processes. This article summarizes contemporary genetic and biochemical strategies that are useful for identifying gene products that have a role in circadian control.

TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior

Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia—different classes of glial cells—have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior.