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The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila Melanogaster

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The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila Melanogaster The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster This information is current as Ashley E. Nazario-Toole, Javier Robalino, Kwame Okrah, of September 27, 2021. Hector Corrada-Bravo, Stephen M. Mount and Louisa P. Wu J Immunol published online 11 July 2018 http://www.jimmunol.org/content/early/2018/07/10/jimmun ol.1800496 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/07/10/jimmunol.180049 Material 6.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 11, 2018, doi:10.4049/jimmunol.1800496 The Journal of Immunology The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster Ashley E. Nazario-Toole,*,† Javier Robalino,* Kwame Okrah,‡ Hector Corrada-Bravo,‡ Stephen M. Mount,*,‡ and Louisa P. Wu*,† The uptake and destruction of bacteria by phagocytic cells is an essential defense mechanism in metazoans. To identify novel genes involved in the phagocytosis of Staphylococcus aureus, a major human pathogen, we assessed the phagocytic capacity of adult blood cells (hemocytes) of the fruit fly, Drosophila melanogaster, by testing several lines of the Drosophila Genetic Reference Panel. Natural genetic variation in the gene RNA-binding Fox protein 1 (Rbfox1) correlated with low phagocytic capacity in hemocytes, pointing to Rbfox1 as a candidate regulator of phagocytosis. Loss of Rbfox1 resulted in increased expression of the Ig superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of Dscam4 in Rbfox1-depleted blood cells rescued the fly’s cellular immune response to S. aureus, indicating that downregulation of Dscam4 by Rbfox1 is critical for S. aureus phagocytosis Downloaded from in Drosophila. To our knowledge, this study is the first to demonstrate a link between Rbfox1, Dscam4, and host defense against S. aureus. The Journal of Immunology, 2018, 201: 000–000. he fruit fly, Drosophila melanogaster, is a powerful ge- In Drosophila, hemocytes play a critical role in host defense netic model for studying host–pathogen interactions. To against bacteria. Adult flies lacking blood cells are unable to T combat infection, the fruit fly solely relies on a robust clear Staphylococcus aureus, a Gram-positive extracellular bac- http://www.jimmunol.org/ innate immune system. The absence of the adaptive immune re- teria (6–8). Similarly, phagocytic blood cells are critical to host sponse in Drosophila facilitates direct in vivo studies of innate defense against S. aureus in humans (8–10). Several hemocyte- immunity. Consequently, studies characterizing the innate defense associated receptors have been shown to be important for reactions of Drosophila have yielded valuable insight into path- S. aureus recognition in the fruit fly: Croquemort (11), scavenger ogen recognition and host defense in higher organisms (1). receptor C1 (12), Eater (13), Nimrod C1 (14), the peptidoglycan The defense reactions of Drosophila include potent humoral and recognition proteins PGRP-SC1A and PGRP-SA (15), Draper cellular responses. Humoral immunity is characterized by the (16), and the integrin heterodimer aPS3/bn (16, 17). With this systemic production of antimicrobial peptides after immune cells large number of receptors, the loss of any given receptor only of the blood and fat body detect bacteria or fungi in the hemo- partially impairs S. aureus uptake. Therefore, the study of phago- by guest on September 27, 2021 lymph. The cellular immune response is carried out by specialized cytosis is complicated by the redundancy of ligand-binding speci- blood cells known as hemocytes, which engulf and eliminate ficities and the overlapping functions of components of the pathogens via phagocytosis (2). During pupal development, phagocytic machinery. phagocytic hemocytes, called plasmatocytes, ingest apoptotic cells In an effort to identify novel genes and signaling pathways and larval tissues (3). After metamorphosis, plasmatocytes adhere involved in the phagocytosis of S. aureus in Drosophila, we un- to heart tissue in the dorsal vessel and serve as the primary effector dertook an approach distinct from traditional forward or reverse of cellular immunity in adult flies (4, 5). genetic screens. We carried out a genome-wide association study using the Drosophila Genetic Reference Panel (DGRP; a panel of D. melanogaster strains with fully sequenced genomes) to find *Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; †Institute for Bioscience and Biotechnology Research, single-nucleotide polymorphisms (SNPs) (18–20) associated with University of Maryland, College Park, MD 20742; and ‡Center for Bioinformatics S. aureus phagocytosis in adult flies. We found that a SNP in RNA- and Computational Biology, University of Maryland, College Park, MD 20742 binding Fox protein 1 (Rbfox1) was present in lines with low ORCID: 0000-0003-2748-8205 (S.M.M.). S. aureus phagocytosis. Received for publication April 4, 2018. Accepted for publication June 14, 2018. Rbfox1 is a member of the highly conserved Fox-1 (Rbfox) This work was supported by a National Science Foundation University of Maryland family of RNA-binding proteins (21–23). Individual Rbfox protein ADVANCE Seed Grant (to S.M.M. and L.P.W.), two National Institutes of Health grants (5T32AI089621 and 5T32GM080201 to A.E.N.-T.), and National Science Foundation isoforms localize either to the nucleus or the cytoplasm of cells Division of Biological Infrastructure Award 1564785 (to co-principal investigator S.M.M). (24–26). Nuclear isoforms regulate tissue-specific alternative The sequences presented in this article have been submitted to the Gene Expression splicing by binding to intronic (U)GCAUG elements in target Omnibus (http://ongen.us/A2BP1-GEOdata) under accession number GSE79488. RNAs, leading to the exclusion of downstream exons or the in- Address correspondence and reprint requests to Dr. Louisa P. Wu, University of clusion of upstream exons (27–31). Recent evidence from studies Maryland, 4112 Plant Sciences Building, College Park, MD 20742-4454. E-mail in Drosophila and humans indicates that cytoplasmic Rbfox1 address: [email protected] isoforms bind to (U)GCAUG elements in the 39 untranslated re- The online version of this article contains supplemental material. gions (UTRs) of specific mRNAs, regulating their stability and Abbreviations used in this article: BH, Benjamini and Hochberg; DE, differential expression; DGRP, Drosophila Genetic Reference Panel; Dscam4, Down syndrome translation (26, 32). In humans, there are three Rbfox family adhesion molecule 4; FDR, false discovery rate; Rbfox1, RNA-binding Fox protein 1; genes: RBFOX1, RBFOX2, and RBFOX3 (23). Disruptions in RNAi, RNA interference; RNAseq, RNA sequencing; RRM, RNA recognition motif; RBFOX1 are associated with neurologic diseases such as mental SNP, single-nucleotide polymorphism; UTR, untranslated region; WT, wild type. retardation, epilepsy, and autism (33, 34). RBFOX2 has been Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 implicated in cancer development and progression as well as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800496 2 RBFOX1 MEDIATES THE CELLULAR IMMUNE RESPONSE IN DROSOPHILA cardiac and neurologic development (35, 36). In Drosophila, of the SNP in Rbfox1 to reflect the most current genome release Rbfox1 is the only Rbfox family member. Rbfox1 has been (FlyBase 6.07). shown to play critical roles in oogenesis and in wing imaginal Larval phagocytosis assay disc development (26, 37, 38). Additionally, Rbfox proteins regulate alternative splicing and transcriptional networks in For each experiment, 10 third-instar wandering larvae were injected with equal volumes of fluorescein-labeled S. aureus (resuspended to 1.6 mg/ml in mouse, human, and Drosophila brains (24, 39–42), indicating sterile PBS with 5% green dye). After 30 min, larvae were bled directly onto that the biological functions of these proteins are conserved polylysine coverslips. After 2 min, carcasses were removed, and the cells across species. were fixed with cold 4% paraformaldehyde in PBS. The fluorescence of In this study, we report a novel role for Rbfox1 in the Dro- bioparticles not taken up by the cells was quenched by briefly washing fixed sophila cellular immune response to Gram-positive bacteria. hemocytes with 5% trypan blue in PBS. The cells were then washed in PBS and the slides were mounted with ProLong with DAPI. Hemocytes were Rbfox1 negatively regulates the expression of the Ig superfamily imaged using a 633 oil immersion lens on a Zeiss LSM 700 confocal mi- member
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