Cell-based assays

2016
Iiro Taneli Helenius, Ryan J Haake, Yong-Jae Kwon, Jennifer A Hu, Thomas Krupinski, Marina S Casalino-Matsuda, Peter HS Sporn, Jacob I Sznajder, and Greg J Beitel. 2016. “Identification of Drosophila Zfh2 as a Mediator of Hypercapnic Immune Regulation by a Genome-Wide RNA Interference Screen..” J Immunol, 2, 196: 655-67, 2016 Jan 15.Abstract

Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse, and Drosophila cells, and it increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA interference improves survival of flies exposed to elevated CO2 and infected with Staphylococcus aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions in Dipt and other antimicrobial peptides and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching, suggesting that Zfh2's role in mediating responses to hypercapnia extends beyond the immune system. Taken together, to our knowledge, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression.

2016_J Immunol_Helenius.pdf Supplement.pdf
Huajin Wang, Michel Becuwe, Benjamin E Housden, Chandramohan Chitraju, Ashley J Porras, Morven M Graham, Xinran N Liu, Abdou Rachid Thiam, David B Savage, Anil K Agarwal, Abhimanyu Garg, Maria-Jesus Olarte, Qingqing Lin, Florian Fröhlich, Hans Kristian Hannibal-Bach, Srigokul Upadhyayula, Norbert Perrimon, Tomas Kirchhausen, Christer S Ejsing, Tobias C Walther, and Robert V Farese. 2016. “Seipin is required for converting nascent to mature lipid droplets..” Elife, 5, 2016 Aug 26.Abstract

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Arunachalam Vinayagam, Meghana M Kulkarni, Richelle Sopko, Xiaoyun Sun, Yanhui Hu, Ankita Nand, Christians Villalta, Ahmadali Moghimi, Xuemei Yang, Stephanie E Mohr, Pengyu Hong, John M Asara, and Norbert Perrimon. 9/13/2016. “An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling.” Cell Reports, 11, 16: 3062-3074, 9/13/2016. Abstract

Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and meta- bolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network sur- rounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47% of interacting proteins affect pathway activity, and, using quantitative phospho- proteomics, we demonstrate that $10% of interact- ing proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by iden- tifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network. 

2016_Cell Rep_Vinayagam.pdf Supplement.pdf
Joel M Swenson, Serafin U Colmenares, Amy R Strom, Sylvain V Costes, and Gary H Karpen. 2016. “The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic..” Elife, 5, 2016.Abstract

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

2016_eLife_Swenson.pdf
Alfeu Zanotto-Filho, Ravi Dashnamoorthy, Eva Loranc, Luis HT de Souza, José CF Moreira, Uthra Suresh, Yidong Chen, and Alexander JR Bishop. 2016. “Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human..” PLoS One, 4, 11: e0153970, 2016.Abstract

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

2016_PLOS One_Zanotto-Filho.pdf Supplemental Files.zip
Stephanie E Mohr. 9/23/2016. “Drosophila research resources at the DRSC and TRiP.” Presentation to the Boston Area Drosophila Meeting (Sept. 2016). 2016_BADM_Mohr.pdf
2015
Stephanie E Mohr, Yanhui Hu, Kirstin Rudd, Michael Buckner, Quentin Gilly, Blake Foster, Katarzyna Sierzputowska, Aram Comjean, Bing Ye, and Norbert Perrimon. 2015. “Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells..” G3 (Bethesda), 9, 5: 1919-24, 2015 Sep.Abstract

RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.

2015_G3_Mohr.pdf Reagent Table S1.xlsx
Richelle Sopko, You Bin Lin, Kalpana Makhijani, Brandy Alexander, Norbert Perrimon, and Katja Brückner. 2015. “A systems-level interrogation identifies regulators of Drosophila blood cell number and survival..” PLoS Genet, 3, 11: e1005056, 2015 Mar.Abstract

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

2015_PLOS Gen_Sopko.pdf Supplemental Files.zip
Joseph Dopie, Eeva K Rajakylä, Merja S Joensuu, Guillaume Huet, Evelina Ferrantelli, Tiao Xie, Harri Jäälinoja, Eija Jokitalo, and Maria K Vartiainen. 2015. “Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators..” J Cell Sci, 13, 128: 2388-400, 2015 Jul 1.Abstract

Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

2015_J Cell Sci_Dopie.pdf Supplement.pdf
Jonathan Zirin, Joppe Nieuwenhuis, Anastasia Samsonova, Rong Tao, and Norbert Perrimon. 2015. “Regulators of autophagosome formation in Drosophila muscles..” PLoS Genet, 2, 11: e1005006, 2015 Feb.Abstract

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

2015_PLOS Genet_Zirin.pdf Supplemental Files.zip
2015. “DRSC: RNA Binding Protein Library S2R+ Baseline Data (data support page for Mohr et al. 2015 "Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells" in G3)”. Publisher's Version
2014
Inma Gonzalez, Julio Mateos-Langerak, Aubin Thomas, Thierry Cheutin, and Giacomo Cavalli. 2014. “Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen..” Mol Cell, 3, 54: 485-99, 2014 May 8.Abstract

Polycomb group (PcG) proteins dynamically define cellular identities through epigenetic repression of key developmental genes. PcG target gene repression can be stabilized through the interaction in the nucleus at PcG foci. Here, we report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. In the absence of SUMO, Pc foci coagulate into larger aggregates. Conversely, loss of function of the SUMO peptidase Velo disperses Pc foci. Moreover, SUMO and Velo colocalize with PcG proteins at PREs, and Pc SUMOylation affects its chromatin targeting, suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci.

2014_Mol Cell_Gonzalez.pdf Supplement.pdf
Dorte Bohla, Martin Herold, Imke Panzer, Melanie K Buxa, Tamer Ali, Jeroen Demmers, Marcus Krüger, Maren Scharfe, Michael Jarek, Marek Bartkuhn, and Rainer Renkawitz. 2014. “A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking..” PLoS One, 9, 9: e107765, 2014.Abstract

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.

2014_PLOS One_Bohla.pdf Supplemental Files.zip
2013
Young Kwon, Arunachalam Vinayagam, Xiaoyun Sun, Noah Dephoure, Steven P Gygi, Pengyu Hong, and Norbert Perrimon. 2013. “The Hippo signaling pathway interactome..” Science, 6159, 342: 737-40, 2013 Nov 8.Abstract

The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.

2013_Science_Kwon.pdf Supplemental Files.zip
Felix Muerdter, Paloma M Guzzardo, Jesse Gillis, Yicheng Luo, Yang Yu, Caifu Chen, Richard Fekete, and Gregory J Hannon. 2013. “A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila..” Mol Cell, 5, 50: 736-48, 2013 Jun 6.Abstract

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

2013_Mol Cell_Muerdter.pdf Supplemental Files.zip
Zheng Yin, Amine Sadok, Heba Sailem, Afshan McCarthy, Xiaofeng Xia, Fuhai Li, Mar Arias Garcia, Louise Evans, Alexis R Barr, Norbert Perrimon, Christopher J Marshall, Stephen TC Wong, and Chris Bakal. 2013. “A screen for morphological complexity identifies regulators of switch-like transitions between discrete cell shapes..” Nat Cell Biol, 7, 15: 860-71, 2013 Jul.Abstract

The way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila haemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous cell populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population, leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.

2013_Nat Cell Bio_Yin.pdf Supplemental Files.zip
Ralph A Neumüller, Thomas Gross, Anastasia A Samsonova, Arunachalam Vinayagam, Michael Buckner, Karen Founk, Yanhui Hu, Sara Sharifpoor, Adam P Rosebrock, Brenda Andrews, Fred Winston, and Norbert Perrimon. 2013. “Conserved regulators of nucleolar size revealed by global phenotypic analyses..” Sci Signal, 289, 6: ra70, 2013 Aug 20.Abstract

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.

2013_Sci Sig_Neumuller.pdf Supplemental Files.zip
Keren Imberg-Kazdan, Susan Ha, Alex Greenfield, Christopher S Poultney, Richard Bonneau, Susan K Logan, and Michael J Garabedian. 2013. “A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells..” Genome Res, 4, 23: 581-91, 2013 Apr.Abstract

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.

2013_Genome Res_Imberg-Kazdan.pdf Supplement.pdf
Clément Carré, Caroline Jacquier, Anne-Laure Bougé, Fabrice de Chaumont, Corinne Besnard-Guerin, Hélène Thomassin, Josette Pidoux, Bruno Da Silva, Eleftheria Chalatsi, Sarah Zahra, Jean-Christophe Olivo-Marin, Hélène Munier-Lehmann, and Christophe Antoniewski. 2013. “AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity..” PLoS One, 9, 8: e74296, 2013.Abstract

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

2013_PLOS One_Carre.pdf Supplemental Files.zip
Clemens Bergwitz, Mark J Wee, Sumi Sinha, Joanne Huang, Charles DeRobertis, Lawrence B Mensah, Jonathan Cohen, Adam Friedman, Meghana Kulkarni, Yanhui Hu, Arunachalam Vinayagam, Michael Schnall-Levin, Bonnie Berger, Lizabeth A Perkins, Stephanie E Mohr, and Norbert Perrimon. 2013. “Genetic determinants of phosphate response in Drosophila..” PLoS One, 3, 8: e56753, 2013.Abstract

Phosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels.

2013_PLOS One_Bergwitz.pdf Supplemental Files.zip

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