Genome-wide screen

2023
Baolong Xia, Raghuvir Viswanatha, Yanhui Hu, Stephanie E Mohr, and Norbert Perrimon. 2023. “Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in cells.” Elife, 12.Abstract

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (), a member of the lipocalin protein family (), and a zinc finger C2H2 transcription factor (). Mechanistically, we demonstrate that overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in cells.

elife-85542-v1.pdf
2022
Ying Xu, Raghuvir Viswanatha, Oleg Sitsel, Daniel Roderer, Haifang Zhao, Christopher Ashwood, Cecilia Voelcker, Songhai Tian, Stefan Raunser, Norbert Perrimon, and Min Dong. 2022. “CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.” Nature, 610, 7931, Pp. 349-355.Abstract
Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Hans M Dalton, Raghuvir Viswanatha, Roderick Brathwaite, Jae Sophia Zuno, Alexys R Berman, Rebekah Rushforth, Stephanie E Mohr, Norbert Perrimon, and Clement Y Chow. 2022. “A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.” PLoS Genet, 18, 9, Pp. e1010430.Abstract
Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.
Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Wei-Chun Tang, Chieh-Han Lu, Stephanie E. Mohr, Robert V. Farese, and Tobias C. Walther. 2022. “Identification of two pathways mediating protein targeting from ER to lipid droplets.” Nature Cell Biol. Publisher's VersionAbstract
Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD–ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.
s41556-022-00974-0-1.pdf
Jonathan Zirin, Justin Bosch, Raghuvir Viswanatha, Stephanie E Mohr, and Norbert Perrimon. 2022. “State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.” Trends Genet, 38, 5, Pp. 437-453.Abstract
For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.
2021
Hans M. Dalton, Raghuvir Viswanatha, Ricky Brathwaite Jr., Jae Sophia Zuno, Stephanie E Mohr, Norbert Perrimon, and Clement Y. Chow. 12/4/2021. “A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress.” BioRxiv. Publisher's VersionAbstract
Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1 CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.
2021.12.03.471178v1.full_.pdf
Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Chieh-Han Lu, Stephanie E. Mohr, Jr Robert V. Farese, and Tobias C. Walther. 9/15/2021. “Identification of two pathways mediating protein targeting from ER to lipid droplets [NOTE: a modified final version was published in Nat Cell Biol and is now available]”. Publisher's VersionAbstract
Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.
2021.09.14.460330v1.full_.pdf
Xiangzhao Yue, Yongkang Liang, Zhishuang Wei, Jun Lv, Yongjin Cai, Xiaobin Fan, Wenqing Zhang, and Jie Chen. 2021. “Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.” Insect Sci.Abstract
Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3(Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr and Abd-A showed the potential complex mechanism of kkv transcription are regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis. This article is protected by copyright. All rights reserved.
2019
Raghuvir Viswanatha, Roderick Brathwaite, Yanhui Hu, Zhongchi Li, Jonathan Rodiger, Pierre Merckaert, Verena Chung, Stephanie E Mohr, and Norbert Perrimon. 2019. “Pooled CRISPR Screens in Drosophila Cells.” Curr Protoc Mol Biol, 129, 1, Pp. e111.Abstract
High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.
2018
Naoki Okamoto, Raghuvir Viswanatha, Riyan Bittar, Zhongchi Li, Sachiko Haga-Yamanaka, Norbert Perrimon, and Naoki Yamanaka. 2018. “A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila.” Dev Cell, 47, 3, Pp. 294-305.e7.Abstract
Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.
2017
Ben Ewen-Campen, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 10/9/2017. “Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR.” Dev Cell, 43, 1, Pp. 6-9.Abstract
Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.
AJ Copeland, A Comjean, N Perrimon, and SE Mohr. 5/15/2017. “Online view of high-content image data generated in the genome-wide screen described in Neumüller et al. 2013, "Conserved regulators of nucleolar size revealed by global phenotypic analyses," made possible using OMERO at HMS”.
2016
Benjamin E Housden, Matthias Muhar, Matthew Gemberling, Charles A Gersbach, Didier YR Stainier, Geraldine Seydoux, Stephanie E Mohr, Johannes Zuber, and Norbert Perrimon. 10/31/2016. “Loss-of-function genetic tools for animal models: cross-species and cross-platform differences.” Nat Rev Genet. Publisher's VersionAbstract

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

2016_Nat Rev Gene_Housden.pdf
Yanhui Hu, Aram Comjean, Charles Roesel, Arunachalam Vinayagam, Ian Flockhart, Jonathan Zirin, Lizabeth Perkins, Norbert Perrimon, and Stephanie E Mohr. 10/11/2016. “FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update.” Nucleic Acids Research. Publisher's VersionAbstract

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.

2016_Nucl Acids Res_Hu.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.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
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, 196, 2, Pp. 655-67.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
2015
Hirotaka Kanoh, Takayuki Kuraishi, Li-Li Tong, Ryo Watanabe, Shinji Nagata, and Shoichiro Kurata. 2015. “Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.” Biochem Biophys Res Commun, 467, 2, Pp. 400-6.Abstract
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.
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, 128, 13, Pp. 2388-400.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
Hirotaka Kanoh, Li-Li Tong, Takayuki Kuraishi, Yamato Suda, Yoshiki Momiuchi, Fumi Shishido, and Shoichiro Kurata. 2015. “Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults.” Sci Signal, 8, 400, Pp. ra107.Abstract
The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling.
J Zanet, E Benrabah, T Li, A Pélissier-Monier, H Chanut-Delalande, B Ronsin, HJ Bellen, F Payre, and S Plaza. 2015. “Pri sORF peptides induce selective proteasome-mediated protein processing.” Science, 349, 6254, Pp. 1356-8.Abstract

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

1356.full_.pdf

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