Cell-based RNAi

Shue Chen, Leah F Rosin, Gianluca Pegoraro, Nellie Moshkovich, Patrick J Murphy, Guoyun Yu, and Elissa P Lei. 8/12/2022. “NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization.” Nucleic Acids Res, 50, 14, Pp. 7906-7924.Abstract
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
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.
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.
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC/TRiP and DRSC-BTRR Office Hours

September 13, 2021

New this fall: Online office hours!

Do you have questions about modifying Drosophila cell lines with CRISPR or performing large-scale cell screens? Questions about in vivo RNAi with TRiP fly stocks or CRISPR knockout or activation with our sgRNA fly stocks? Questions about our new protocols and resources for CRISPR mosquito cell lines? Pop into our Zoom office hours to say hello and get our expert input! Registration is required (see below).

DRSC/TRiP & DRSC-BTRR Office Hours Schedule:

Mon. Sept. 27, 2021, 12...

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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.
Yanhui Hu, Aram Comjean, Jonathan Rodiger, Yifang Liu, Yue Gao, Verena Chung, Jonathan Zirin, Norbert Perrimon, and Stephanie E Mohr. 2020. “FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update.” Nucleic Acids Res.Abstract
The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) provides a suite of online resources that facilitate functional genomics studies with a special emphasis on Drosophila melanogaster. Currently, the database provides: gene-centric resources that facilitate ortholog mapping and mining of information about orthologs in common genetic model species; reagent-centric resources that help researchers identify RNAi and CRISPR sgRNA reagents or designs; and data-centric resources that facilitate visualization and mining of transcriptomics data, protein modification data, protein interactions, and more. Here, we discuss updated and new features that help biological and biomedical researchers efficiently identify, visualize, analyze, and integrate information and data for Drosophila and other species. Together, these resources facilitate multiple steps in functional genomics workflows, from building gene and reagent lists to management, analysis, and integration of data.
Graphical image of tissue culture, fly pushing, and computer, and the team of people who work with them

DRSC-Biomedical Technology Research Resource

October 21, 2019

We are pleased to announce that we have been funded by NIH NIGMS to form the Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR). The P41-funded DRSC-BTRR (N. Perrimon, PI; S. Mohr, Co-I) builds upon and extends past goals of the Drosophila RNAi Screening Center.

As the DRSC-BTRR, we are working together with collaborators whose 'driving biomedical projects' inform development of new technologies at the DRSC. At the same time, we continue to support Drosophila cell-based RNAi and CRIPSR knockout screens and related...

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Drosophila cell screen with DRSC reagent library contributes to identification of new therapeutic target for renal cancer

October 7, 2019

We here at the DRSC/TRiP are thrilled to see this study from Hilary Nicholson et al. published in Science Signaling.

The study provides a great example of how screens in Drosophila cultured cells can be used as part of a cross-species platform aimed at discovery of new targets for disease treatment. The work represents a collaboration between the laboratory of 2019 Nobel Prize winner W. Kaelin and DRSC PI N. Perrimon.

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Hilary E Nicholson, Zeshan Tariq, Benjamin E Housden, Rebecca B Jennings, Laura A Stransky, Norbert Perrimon, Sabina Signoretti, and William G Kaelin. 2019. “HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species.” Sci Signal, 12, 601.Abstract
Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.
Cartoon of fly host cells with virus or endosymbiotic bacteria

Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports

November 19, 2018

Laboratories at the Skirball Institute at New York University and the Boyce Thompson Institute at Cornell University reported results of two different cell-based Drosophila RNAi screens in papers published this week. The screens have in common that they looked at interactions between the host insect cells and a microbe -- the endosymbiont Wolbachia in one study and baculovirus in the other. For more, check out the newly published studies. For both these screens, the DRSC provided libraries for screens that were then performed at the host institution.

 

... Read more about Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports
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.
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.

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