Sung and Shears BMC Res Notes (2018) 11:884 https://doi.org/10.1186/s13104-018-3996-z BMC Research Notes RESEARCH NOTE Open Access A genome‑wide dsRNA library screen for Drosophila genes that regulate the GBP/ phospholipase C signaling axis that links infammation to aging Eui Jae Sung and Stephen B. Shears* Abstract Objective: Invertebrates are productive models for understanding how infammation, metabolism and aging are intertwined. We have deployed a dsRNA library screen to search for genes in Drosophila melanogaster—and hence 2 identify human orthologs—that encode participants in a G-protein coupled, Ca­ +-signaling pathway that regulates infammation, metabolism and lifespan. 2 Results: We analyzed receptor-dependent, phospholipase C/Ca + signaling responses to the growth-blocking peptide (GBP) cytokine in Drosophila S3 cells plated in 384-well plates containing dsRNAs that target approximately 14,000 Drosophila genes. We used Z-scores of < 3 or > 3 to defne gene hits. Filtering of ‘housekeeping’ genes − + 2 from these hits yielded a total of 82 and 61 Drosophila genes that either down-regulate or up-regulate Ca­ +-signaling, respectively; representatives from these two groups were validated. Human orthologs of our hits may be modulators 2 of ­Ca + signaling in general, as well as being candidates for acting in molecular pathways that interconnect aging and infammation. Keywords: Cytokine, Infammation, Metabolism, Calcium-signaling, G-proteins, Receptor Introduction Invertebrates are productive, genetically-tractable A systems-level understanding of cytokine-mediated, models for understanding how infammation and aging inter-tissue signaling can help to generate fundamen- are inter-related in humans [1, 4]. Recent work has estab- tal insight into links between longevity, metabolism and lished that a Drosophila cytokine, growth-blocking pep- infammation [1]. Research with human subjects indi- tide (GBP), interconnects longevity, infammation and cates that aging is afected by the balance between cir- dietary infuences, through activation of the phospho- culating pro- and anti-infammatory factors [2] which lipase C (PLC)/Ca2+ signaling cascade [5, 6]. Te spati- are subject to various environmental infuences, among otemporal control of stimulus-activated ­Ca2+ dynamics which calorifc restriction receives particular atten- by PLC is a multiplex cellular process that coordinates tion [3]. However, little is known concerning the precise three fundamental activities: maintenance of basal ­Ca2+ nature of the molecular entities and pathways that inter- pools, release of Ca­ 2+ from intracellular stores, and twine these biological phenomena in humans; most work ­Ca2+ fuxes across the plasma membrane [7]. A com- in this area uses animal models. plete understanding of the integration of these processes requires characterization of the networking of a multi- tude of individual regulatory components; this degree of *Correspondence: [email protected] systems-biology insight has not yet been attained. Some Inositol Signaling Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, important information has been obtained by exploit- 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA ing the amenability of Drosophila to the application of © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sung and Shears BMC Res Notes (2018) 11:884 Page 2 of 7 dsRNA libraries [8, 9]. However, the latter studies were unusual, ‘negative’ gene-hits: dsRNAs that yielded an 2+ technically limited to identifcation of the subset of pro- increase in [Ca ]T (with a Z-score greater than 3). teins that impact Ca 2+ fuxes across the plasma mem- We encountered more plate-to-plate variability than in brane; in the current study, we have sought to widen the our previous study with the transmembrane dsRNA sub- knowledge-base through identifcation of regulatory fac- library [5]. In the current study, for a number of plates, 2+ 2+ tors that may impact release of Ca release from intra- the cells in individual wells exhibited values for [Ca ]T cellular stores and Ca2+ fuxes into the cell, i.e., total Ca2+ that clustered atypically close to that of the control, rais- 2+ mobilization ([Ca ]T). ing the possibility that knock-down efciencies were For this work we deployed a fuorophore-based assay abnormally low. We did not establish the cause of this for GBP-mediated Ca2+ mobilization in Drosophila S3 variability, although it was not specifc to any particular cells in a 384-well, high-throughput screening format [5]. plate barcodes. We resolved this problem in the following In that latter study, we screened a relative small dsRNA manner: the Itp-r83A gene was one of our initial, and sta- sub-library that targets 1729 genes known (or computa- tistically most signifcant hits (Table 1); we independently tionally predicted) to encode transmembrane proteins. validated this hit using alternate dsRNA constructs In that way, we identifed several integral membrane pro- (Fig. 1a). Tus, we decided to use this as an internal teins that contribute to GBP-mediated Ca2+ release [5]. control. Tis led us to establish that an adequate range 2+ Nevertheless, that particular dsRNA sub-library only of [Ca ]T signals within a plate was typically observed covered 12% of all Drosophila genes. In the current study, whenever our Itp-r83A dsRNA caused at least 60% inhi- we performed a genome-wide dsRNA screen (almost bition of GBP-mediated Ca2+ signaling. Tus, all data 14,000 genes) so as to identify a more complete set of were discarded from any plate in which the inhibition by regulatory factors. Tis approach could be particularly the internal Itp-r83A dsRNA control did not reach the benefcial in the context of diseases where changes in 60% cut-of. We were able to procure new plates from Ca2+-signaling are causative, since that can provide iden- the vendor to replace most of those plates that we dis- tify novel therapeutic targets [7]. carded, and eventually we performed enough assays to screen every dsRNA in the genome-wide library at least Main text once. Previous studies that used similar plates to screen Methods for gene knock-downs that target Ca 2+ entry into the cell Screening of the dsRNA library [8, 9] do not state if a similar problem was experienced. We purchased a genome-wide dsRNA library (v2.0) from However, a diferent version of the dsRNA library (i.e., the DRSC/TRiP Functional Genomics Resources (https v1) was deployed in those earlier studies; we used v2. ://fgr.hms.harva rd.edu/). Tis library contains 66 × 384 Te complete list of Z-scores is available at http:// well-plates that target almost 14,000 Drosophila genes www.fyrn ai.org/. Here, we tabulate two fltered hit-lists with either one or two dsRNA constructs. Te library is (Tables 1 and 2), from which we have removed all hits provided in duplicate (i.e., as a total of 132 plates). Te that could reasonably be predicted to non-specifcally biological readout during the screening was the total, afect protein synthesis, by virtue of their having the GBP-induced change in fuorescence (equivalent to following categorizations in the Gene Ontology Data- 2+ 2+ [Ca ]T) emitted from a genetically encoded Ca sen- base (http://www.geneo ntolo gy.org/): mRNA splic- sor in Drosophila S3 cells, in response to the addition of ing (GO:0000398); structural constituent of ribosome 500 nM GBP (added after 5-days pre-treatment with the (GO:0003735); translation initiation factor activity dsRNAs [5]. A FLIPRTETRA (Molecular Devices) was used (GO:0003743); transcription factor activity, sequence- 2+ to simultaneously record [Ca ]T in every well of a 384- specifc DNA binding (GO:0003700); RNA polymerase well plate. We describe these procedures in more detail II transcription cofactor activity (GO:0001104); RNA in our previous study with a small dsRNA sub-library polymerase II activity (GO:0001055); DNA-directed that targets 1729 genes [5]. RNA polymerase activity (GO:0003899); transcription factor binding (GO:0008134); transcriptional activa- Evaluation and deposition of the screening data tor activity, RNA polymerase II core promoter proximal Z-scores were calculated for every unique dsRNA. A region sequence-specifc binding (GO:0001077); transla- ‘positive’ gene-hit for any single dsRNA was defned tion (GO:0006412); translation elongation factor activity 2+ by a reduction in [Ca ]T that yielded a Z-score of less (GO:0003746). 2+ than − 3; the Z-score of a dsRNA = ([[Ca ]T of each 2+ Independent validation and additional screening dsRNA] − [average [Ca ]T of plate])/[SD of plate 2+ 2+ [Ca ]T]. Tis strict disambiguation approach is designed Follow-up GBP-mediated Ca mobilization in S3 cells, to avoid false positives. Additionally, we describe some using unique dsRNA constructs diferent from those used Sung and Shears BMC Res Notes (2018) 11:884 Page 3 of 7 Table 1 Filtered list of gene knock-downs that reduced Table 1 (continued) GBP-mediated