Project description:Circadian behaviors are regulated by intrinsic biological clocks consisting of central molecular oscillators and output pathways. Despite significant progress in elucidating the central timekeeping mechanisms, the molecular pathways coupling the circadian pacemaker to overt rhythmic behavior and physiology remain elusive. The Drosophila LARK RNA-binding protein is a candidate for such a coupling factor. Previous research indicates that LARK functions downstream of the clock to mediate behavioral outputs. To better understand the roles of LARK in the Drosophila circadian system, we sought to identify RNA molecules associated with LARK in vivo, using a novel strategy that involves capturing the RNA ligands by immunoprecipitation, visualizing the captured RNAs using whole gene microarrays, and identifying functionally relevant targets through genetic screens. Experiment Overall Design: LARK-containing ribonucleoprotein complexes (LARK-RNPs) were precipitated from lysates of hand-dissected pharate adult brains using an affinity-purified anti-LARK antibody (around 1000 brains were used per immunoprecipitation experiment). A portion of each lysate was saved prior to immunoprecipitations (IPs) in order to measure the relative abundance of transcripts in a total RNA sample. RNAs extracted from the LARK-RNP and total RNA samples were labeled and hybridized to Drosophila whole-genome gene microarrays; signal intensities for individual genes were compared between samples to identify those RNAs that were enriched by immunoprecipitation (relative to their abundances in total RNA). RNAs that were selectively enriched in the LARK-RNP samples were considered to be potential targets of the RNA-binding protein. Experiment Overall Design: Due to the difficulty to dissect large amount of fly brains, only two such immunoprecipitation experiments were performed, each generating an IP RNA sample and a total RNA (control) sample. The amount of RNAs obtained from IP is very small thus only one array is used for each sample - i.e. there are only biological replicates and no technical replicate.

Project description:While microRNAs (miRNAs) have recently emerged as critical post-transcriptional modulators of gene expression in neuronal development, very little is known regarding the roles of miRNA-mediated regulation in the specification of cell-type specific dendritic complexity. The dendritic arborization (da) sensory neurons of the Drosophila PNS offer an excellent model system for elucidating the molecular mechanisms governing class specific dendrite morphogenesis and for exploring miRNA-mediated control of this process. To facilitate functional analyses of miRNA regulation in da neurons, we have conducted whole-genome miRNA expression profiling as well as mRNA expression profiling of three distinct classes of da neurons, thereby generating a comprehensive molecular gene expression signature within these individual subclasses of da neurons. To further validate the role of these expressed miRNAs in directing dendritic architecture, we conducted a genome-wide UAS-miRNA phenotypic screen using live-image confocal microscopy followed by semi-automated neurometric quantification, to directly assess the effect of over/mis-expression of individual and clustered miRNAs on neurons of varying dendritic complexity. Through this approach, we have identified numerous miRNAs with previously unknown functions in dendritic development. Gain-of-function and loss-of-function analyses revealed an endogenous role miR-2b and miR-13b (members of the K-box family) and miR-12/283/304 in dendritic patterning in da neuron subclasses. Moreover, using an integrative bioinformatic analysis approach involving inverse correlation between miRNA and mRNA expression profiling data in combination with existing target prediction algorithms, we have identified putative target of these miRNAs in regulating da neuron dendritic development. Validation of these predicted miRNA-target relationships via phenotypic analyses as well as QPCR, revealed the regulatory effect of these molecules in restricting dendritic branching in da neurons.

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Drosophila melanogaster

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