Research ArticleImmunology

Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults

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Science Signaling  27 Oct 2015:
Vol. 8, Issue 400, pp. ra107
DOI: 10.1126/scisignal.2005971
  • Fig. 1 Properties of DL1 cells in Toll pathway activation and validation of Toll and Imd signaling efficiency in the genome-wide RNAi screenings.

    (A) Luciferase reporter assays monitoring the activation of the Drosomycin promoter were performed in the indicated cell lines, which were transfected with the Drs-luc reporter plasmid and with expression plasmids encoding either dMyd88 or Toll-ΔLRR, as indicated. (B) Relative luciferase activity (RLA) was measured in DL1 cells transfected with an expression plasmid encoding Toll-ΔLRR together with plasmids encoding dsGFP or dsDif. (C and D) S2R and DL1 cells were transfected with increasing amounts of plasmids encoding Toll-ΔLRR (C) or PGN-EK (D) and then were subjected to luciferase assays to compare the extents of Toll signaling (C) and Imd signaling (D). (E to H) RLA was assessed in DL1 cells transfected with plasmids encoding dMyd88 (E), Pelle (F), or Dif (G) for overexpression or encoding Cactus-specific double-stranded RNA (dsRNA) for knockdown (H) together with the indicated dsRNAs. dsGFP was used as a negative control. Data in (A) to (H) are means ± SEM of triplicate wells from a single experiment and are representative of two independent experiments. *P < 0.05 by Student’s t test. (I) Concept behind the comparative genome-wide RNAi screen. Four distinct stimuli and the putative functions of target genes are represented in the signaling map of the Toll pathway. (J) Heat map of the RLA in cells overexpressing or deficient in known signaling components in the Toll and Imd pathway. The percentage RLA relative to the median is shown with a colored background. Red indicates 20% of median RLA, whereas black and green indicate changes in the range from 60 to 180% of the median RLA. More than one dsRNA amplicon was used for those genes that appear more than once.

  • Fig. 2 Identification of possible ubiquitin- and SUMO-related factors and protein kinases.

    (A) Heat map comparison of the RLA values of cells transfected with dsRNAs targeting the E3 ubiquitin ligases encoded by CG9153 and CG42593 and the SUMO homolog encoded by Smt3 in each screen (single replica) and those of cells transfected with dsRNAs targeting other E2 and E1 enzymes and ubiquitin-like modifiers. (B and C) Gene symbols of candidate genes encoding protein kinases that satisfied the criteria of each screen are indicated and compared in the Venn diagram. (D) Heat map comparison of the RLA values of cells in each screen transfected with dsRNAs targeting the indicated candidate kinase-encoding genes. In the heat map, red indicates a decrease in RLA to 20%, whereas black and green indicate no change in the range from 60 to 180%. More than one dsRNA amplicon was used for those genes that appear more than once in the list.

  • Fig. 3 Sherpa is required for host defense against Gram-positive bacterial and fungal infections.

    (A to F) The percentage survival rates of sherpa1 flies injected with saline (A and D), Staphylococcus saprophyticus (B and F), Enterococcus faecalis (C), and Ecc15 (E) were compared with those of yellow white (yw) flies and sherpaRev files, as well as with flies with mutations in either the Toll pathway (spzrm7) or the Imd pathway (RelE20). Data are representative of two (A and D) or more than three (B, C, E, and F) independent experiments with ~30 flies in each experiment. *P < 0.05 by log-rank test. (G to N) Analysis of the expression of genes encoding antimicrobial peptides in sherpa mutants. The relative abundances of Drosomycin (G to L) and Diptericin (M and N) mRNAs were determined by qPCR analysis of total RNA extracted from wild-type, spzrm7, RelE20, sherpa1, sherpaRev, c564>lacZ (UAS-Dcr2/+;c564-GAL4/UAS-lacz), c564>Toll-RNAi (UAS-Dcr2/+;c564-GAL4/UAS-Toll-RNAi), and c564>sherpa-RNAi (UAS-Dcr2/+;c564-GAL4/UAS-sherpa-RNAi) flies. The RNA samples were collected at 24 hours (G to I, K, and L), 18 hours (J), or 6 hours (M and N) after the flies were injected with saline (G), S. saprophyticus (H and L), E. faecalis (I), Candida albicans (J), PGN-SA (peptidoglycan from Staphylococcus aureus) (K), Ecc15 (M), or PGN-EK (peptidoglycan from Escherichia coli) (N). Data in (G) to (N) are representative of more than three independent experiments each performed in triplicate (10 flies ×3). *P < 0.05 by Student’s t test. Or, Oregon R strain; ns, not significant.

  • Fig. 4 Sherpa functions genetically upstream of Pelle and Tube in DL1 cells.

    (A to D) DL1 cells were transfected with Drs-luc together with empty plasmid (Cntl) or expression plasmids to overexpress the indicated combinations of proteins and dsRNAs before being subjected to luciferase reporter assays. (E) Schematic representation of Toll signaling and the genetic interaction of sherpa. (F) DL1 cells were transfected with the Drs-luc reporter and plasmid expressing dMyd88 together with plasmids encoding full-length Sherpa (Sherpa-FL), Sherpa-ΔHECT, or Sherpa-ΔRCC, as well as with dsRNA targeting GFP or the 5′UTR of Sherpa before being analyzed to determine luciferase activity. (G) Schematic representation of the protein structure of Sherpa. Seven RCC repeats whose function is unknown reside in the N terminus of Sherpa, and the HECT domain required for its E3 ubiquitin ligase activity is located at the C terminus of the protein. The 3×HA tag was added to the C terminus of Sherpa. Mutant Sherpa proteins devoid of the HECT domain or the RCC repeat are shown. Data in (A) to (D) and (F) are means ± SEM of triplicate samples from a single experiment and are representative of two (D), three (B, C, and F), and four (A) independent experiments. *P < 0.05 by Student’s t test.

  • Fig. 5 Detection of physical interactions between dMyd88 and Sherpa and of posttranslational modifications, including polyubiquitylation.

    (A) DL1 cells were transfected with plasmid encoding dMyd88-V5, plasmid encoding Sherpa-3×HA, or both, and whole-cell lysates were subjected to immunoprecipitation (IP) with anti-V5 (middle) or anti-HA (right) antibodies, and both lysates and immunoprecipitated samples were subjected to Western blotting (IB) analysis with antibodies specific for the indicated proteins or tags. The polyubiquitylation of target proteins was detected with an anti-ubiquitin (Ub) antibody and a K63 linkage–specific antibody. (B) DL1 cells transfected with control plasmid or with expression plasmids encoding 3×HA-tagged Sherpa-FL, Sherpa-ΔHECT, or Sherpa-ΔRCC were subjected to immunoprecipitation with an anti-HA antibody, and immunoprecipitated samples and lysates were analyzed by Western blotting with antibodies against the indicated proteins. (C) DL1 cells transfected with control plasmid or with expression plasmid encoding dMyd88-V5 were cotransfected with expression plasmids encoding 3×HA-tagged Sherpa-FL, Sherpa-ΔHECT, or Sherpa-ΔRCC, as indicated. Cell lysates were then subjected to immunoprecipitation with an anti-V5 antibody, and immunoprecipitated samples and lysates were analyzed by Western blotting with antibodies against the indicated proteins. All Western blots are representative of at least two independent experiments. MW, molecular weight; HMW, high molecular weight.

  • Fig. 6 Sherpa and Smt3 are required for the plasma membrane localization of the dMyd88-Tube complex in DL1 cells.

    (A) DL1 cells were transfected with plasmids encoding 3×HA-tagged Sherpa-FL, Sherpa-ΔHECT, or Sherpa-ΔRCC, and the subcellular localization was determined by confocal microscopy. Sherpa proteins were visualized with an anti-HA antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 100 μm. Images are representative of three independent experiments. (B) The percentages of the cells represented in (A) that had the indicated Sherpa constructs localized to the plasma membrane as compared to the cytosol were quantified. Data are means ± SEM of three independent experiments. *P < 0.05 by Student’s t test. (C) DL1 cells cotransfected with expression plasmids encoding FLAG-tagged dMyd88 and V5-tagged Tube proteins were also transfected with dsGFP (control), dsSherpa, or dsSmt3. The cells were then analyzed by confocal microscopy to determine the subcellular distributions of dMyd88 (red, top) and Tube (green, bottom). Scale bar, 100 μm. Images are representative of three independent experiments. (D) The percentages of the cells represented in (C) that showed plasma membrane localization of the dMyd88-Tube complex were quantified for each condition. Data are means ± SEM of three independent experiments. *P < 0.05 by Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/400/ra107/DC1

    Fig. S1. Numbers of candidate genes for each screening sorted by functional annotations.

    Fig. S2. Expression profiles of sherpa and the sherpa1 loss-of-function mutant.

    Fig. S3. Knockdown efficiency of sherpa and smt3 in DL1 cells.

    Fig. S4. Quantification of coimmunoprecipitated proteins and posttranslational modifications, including polyubiquitylation.

    Fig. S5. Western blotting analysis of Tube coexpressed with dMyd88 and Smt3.

    Fig. S6. Expression of Drosomycin and Diptericin mRNAs in Pitslre-RNAi flies and analysis of Drosomycin promoter activity in Doa-RNAi cells.

    Table S1. Sequences of DNA primers used for dsRNA synthesis.

    Data file S1. List of candidate genes for each screening sorted by functional annotation.

  • Supplementary Materials for:

    Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults

    Hirotaka Kanoh, Li-Li Tong, Takayuki Kuraishi,* Yamato Suda, Yoshiki Momiuchi, Fumi Shishido, Shoichiro Kurata*

    *Corresponding author. E-mail: akayuki.kuraishi{at}gmail.com (T.K.); kurata{at}mail.pharm.tohoku.ac.jp (S.K.)

    This PDF file includes:

    • Fig. S1. Numbers of candidate genes for each screening sorted by functional annotations.
    • Fig. S2. Expression profiles of sherpa and the sherpa1 loss-of-function mutant.
    • Fig. S3. Knockdown efficiency of sherpa and smt3 in DL1 cells.
    • Fig. S4. Quantification of coimmunoprecipitated proteins and posttranslational modifications, including polyubiquitylation.
    • Fig. S5. Western blotting analysis of Tube coexpressed with dMyd88 and Smt3.
    • Fig. S6. Expression of Drosomycin and Diptericin mRNAs in Pitslre-RNAi flies and analysis of Drosomycin promoter activity in Doa-RNAi cells.
    • Table S1. Sequences of DNA primers used for dsRNA synthesis.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). List of candidate genes for each screening sorted by functional annotation.

    Citation: H. Kanoh, L.-L. Tong, T. Kuraishi, Y. Suda, Y. Momiuchi, F. Shishido, S. Kurata, Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults. Sci. Signal. 8, ra107 (2015).

    © 2015 American Association for the Advancement of Science

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