A Genetic Screen for Localized mRNAs and Investigation of a Selected Positive Hit in Drosophila Class IV da Neurons

mRNA localization is important for polarized cells in that it spatially restricts gene expression and allows for rapid response to local stimuli through on-site regulation of translation. This phenomenon is well-characterized in a variety of organisms and cell types, but its role during neuronal dendritic development is not yet fully understood. Further research into this area could have implications for a variety of neurodevelopmental and psychiatric disorders associated with dendritic abnormalities, such as autism and schizophrenia. Here, we conducted a screen to identify mRNA transcripts that localize to the processes of class IV dendritic arborization (da) neurons in Drosophila melanogaster third instar larvae. These are sensory neurons with highly elaborated dendritic arbors reminiscent of some types of cortical neurons in mammals. The screen was conducted by randomly tagging endogenous mRNA transcripts in vivo with MS2 RNA stem-loops, which bind MS2 coat protein-red fluorescent protein (MCP-RFP fusion protein) to enable subsequent visualization of transcript localization by fluorescence confocal microscopy. Twenty-seven percent of the lines screened exhibited mRNA localization to axons and dendrites. Two of these genes were identified via TAIL-PCR as Sir2 and Mrp4. The role of Sir2 in class IV da neurons was investigated by targeted knockdown and overexpression. The author found that Sir2 overexpression results in a more forceful response to noxious stimuli and in defective foraging locomotion relative to control. Ultimately, these data lay the groundwork for future studies examining the role of mRNA localization in the regulation of gene function in neurons

A genome-wide screen for dendritically localized RNAs identifies genes required for dendrite morphogenesis.

Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling

A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis

Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling

Somatic mosaicism and common genetic variation contribute to the risk of very-early-onset inflammatory bowel disease

Very-early-onset inflammatory bowel disease (VEO-IBD) is a heterogeneous phenotype associated with a spectrum of rare Mendelian disorders. Here, we perform whole-exome-sequencing and genome-wide genotyping in 145 patients (median age-at-diagnosis of 3.5 years), in whom no Mendelian disorders were clinically suspected. In five patients we detect a primary immunodeficiency or enteropathy, with clinical consequences (XIAP, CYBA, SH2D1A, PCSK1). We also present a case study of a VEO-IBD patient with a mosaic de novo, pathogenic allele in CYBB. The mutation is present in ~70% of phagocytes and sufficient to result in defective bacterial handling but not life-threatening infections. Finally, we show that VEO-IBD patients have, on average, higher IBD polygenic risk scores than population controls (99 patients and 18,780 controls; P < 4 × 10-10), and replicate this finding in an independent cohort of VEO-IBD cases and controls (117 patients and 2,603 controls; P < 5 × 10-10). This discovery indicates that a polygenic component operates in VEO-IBD pathogenesis