Research ArticleGENE REGULATION

Stratified ubiquitination of RIG-I creates robust immune response and induces selective gene expression

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Science Advances  22 Sep 2017:
Vol. 3, no. 9, e1701764
DOI: 10.1126/sciadv.1701764
  • Fig. 1 A stratified mechanism of RIG-I–N multisite ubiquitination.

    (A) Schematic map for reported conjugated ubiquitination sites on RIG-I–N. (B) Luciferase activity in RIG-I KO 293T cells (RIG-INull) transfected with an interferon-stimulated response element (ISRE) luciferase (ISRE-luc) reporter, together with an empty vector (EV), RIG-IWT, or RIG-I8KR, followed by treatment with or without IC poly(I:C) [low molecular weight (LMW); 5 mg/ml; the same dosage will be used if there is no special note]. Immunoblot was performed using anti-Flag antibodies to measure the expression of RIG-I in RIG-IWT– and RIG-I8KR–transfected RIG-INull 293T cells. (C) Immunoblot (IB) analysis of extracts of RIG-INull cells transfected with RIG-I WT or its mutants and treated with or without IC poly(I:C) for 12 hours. (D) Re-coimmunoprecipitation (Re-IP) and immunoblot analysis of extracts of 293T cells transfected with RIG-I WT or 8KR, together with hemagglutinin (HA)–K63 ubiquitin (Ub), with or without IC poly(I:C) treatment. Note: Samples after one-time immunoprecipitation (IP) were boiled for 5 min with 10% SDS. The supernatant of samples was then diluted 10 times and used for second-time immunoprecipitation with anti-Flag beads. WCL, whole-cell lysate. (E) Transcriptome sequencing analysis of WT, RIG-INull, and RIG-I8KR A549 cells treated with SeV for indicated time points. The fragments per kilobase million (FPKM) values of the top 200 up-regulated genes were shown. (F) Luciferase activity in RIG-INull cells transfected with ISRE-luc, together with EV, RIG-I WT, DM, or 6KR, followed by treatment with or without IC poly(I:C). Immunoblot was performed using anti-Flag antibody to quantify the expression of RIG-IWT and mutants. (G) Immunoblot analysis of extracts of RIG-INull cells transfected with RIG-I WT or its mutants and treated with or without IC poly(I:C) for 12 hours. (H) Re-coimmunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with RIG-I WT, DM, or 6KR, together with HA-K63 ubiquitin, with or without IC poly(I:C) treatment for 12 hours. (I) Luciferase activity in RIG-INull cells transfected with ISRE-luc, together with EV, RIG-I WT, or its mutants, followed by treatment with or without IC poly(I:C). Immunoblot at the bottom showed equal expression of RIG-I WT and its various mutants. (J) Immunoblot analysis of extracts of RIG-INull cells transfected with RIG-I WT or its mutants and treated with or without IC poly(I:C) for 12 hours. (K) Re-coimmunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with RIG-I WT or indicated mutants, together with HA-K63 ubiquitin, with or without IC poly(I:C) treatment for 12 hours. (L) Illustration of stratified RIG-I ubiquitination process. RIG-I in green, inactivated state; RIG-I in red, active state. Data in (B), (F), and (I) are means ± SD of three independent experiments. ***P < 0.001 versus RIG-IWT cells.

  • Fig. 2 RIG-I–N multisite ubiquitination controls the ultrasensitivity of RIG-I–mediated type I IFN signaling activation.

    (A) Fluorescence microscope analyses of 293T cells transfected with RIG-I–eGFP with or without IC poly(I:C) for 6 hours. Bright spots were measured by ImageJ. Scale bars, 10 μm. (B) Dose-response curves [10−4 (−4), 10−3 (−3), 10−2 (−2), 10−1 (−1), 100 (0), and 101 (1) μg/ml] in indicated cell lines followed by treatment with or without IC poly(I:C). Each point represents the mean value of the bright spot area from 50 cells. (C) Dose responses [10−4 (−4), 10−3 (−3), 10−2 (−2), 10−1 (−1), 100 (0), and 101 (1) μg/ml] in indicated cell lines transfected with ISRE-luc, followed by treatment with or without IC poly(I:C). Each point represents four independent experiments. ISRE activity was normalized by the maximum activation level. (D) Mathematical model of RIG-I ubiquitination. The subscripts in R denote the ubiquitination state, where R represents RIG-I. In the initiation phase, either K164 or K172 was randomly ubiquitinated. After initiation, the ubiquitin chains were further randomly ligated on RIG-I and resulted in ubiquitination amplification. “0”: without ubiquitin; “1”: ubiquitinated. (E) Violin plots for Hill coefficients of RIG-I oligomerization (top) and ISRE activation (bottom) through computational simulation. The violin plots were based on kernel density estimation using the ksdensity function in MATLAB. n = 1000 sets for each condition. The green circles represent the experimental data in (B) and (C). (F) Simulation of ISRE-luc activity. (Top) Statistical significance can be detected for any two groups except that between the K164R and K172R groups (P = 0.7462). (Bottom) Function of RIG-I can be awakened by either of the two initiation sites cooperating with the other six noncore sites. a.u., arbitrary unit. (G) Partial rank correlation coefficient (PRCC) for parameters and nonzero initial conditions. Data in (B) and (C) are means ± SD of at least three independent experiments.

  • Fig. 3 Multisite ubiquitination of RIG-I orchestrates the robustness and ultrasensitivity of type I IFN signaling activation.

    (A) The significantly up-regulated (Up) and down-regulated (Down) groups were marked with dark blue points. The expression with P < 0.05 and at least twofold changes was chosen. Horizontal dashed line indicates P = 0.05, whereas the vertical dashed lines represent the expression with twofold change. Ratio, WT/6KR. (B) Pairwise comparison among the log2FC of the top 2000 up-regulated genes in the indicated four cell lines. (C) Gene clusters in four cell lines. n, number of genes in the indicated pattern. (D to F) Real-time PCR analysis of IFNB1 and indicated ISGs in A549 RIG-IWT, RIG-IK164R, RIG-IK172R, and RIG-I6KR cells treated with SeV for indicated time points. (D) Genes specifically up-regulated by high intensity of type I IFNs. (E) Genes specifically up-regulated by high ultrasensitivity of type I IFN signaling. (F) IFNB1 mRNA level and genes not affected by IFN intensity and ultrasensitivity. (G) CV for RIG-I oligomerization under different conditions under viral stimulation. Data in (D) to (F) are means ± SD of three independent experiments. NS, not significant. *P < 0.05 and ***P < 0.001 versus RIG-IWT cells.

  • Fig. 4 E3 ligases of RIG-I determine the specific ISG expression by shaping the IFN activation intensity and ultrasensitivity.

    (A) Schematic of RIG-I ubiquitination sites regulated by different E3 ligases. (B) Hill coefficient distributions for WT (blue), TRIM4 KO (yellow), TRIM25 KO (gray), and MEX3C KO (green) conditions via computational simulation. n = 1000 for each. Statistical significance was detected between TRIM4 KO and TRIM25 KO (P = 0.0029) or MEX3C KO and TRIM25 (P < 0.0001) groups using Mann-Whitney test. (C and D) Dose responses [10−4 (−4), 10−3 (−3), 10−2 (−2), 10−1 (−1), 100 (0), and 101 (1) μg/ml (C) or multiplicity of infection (MOI) (D)] of luciferase activity in RIG-IWT 293T cells transfected with an ISRE-luc reporter, together with control siRNA (NC), TRIM4-specific siRNA (siTRIM4), TRIM25-specific siRNA (siTRIM25), or MEX3C-specific siRNA (siMEX3C), followed by treatment with or without IC poly(I:C) (C) or SeV (D). Each point represents four independent experiments. ISRE activity was normalized by the maximum level. (E) Real-time PCR for IFNB1 and indicated ISGs in A549 RIG-IWT cells transfected with NC or siTRIM25, followed by treatment with SeV for the indicated duration. Data in (C) and (D) are means ± SD of at least three independent experiments. *P < 0.05 and ***P < 0.001 versus cells transfected with NC siRNA.

  • Fig. 5 Different RIG-I ubiquitination modes generate diverse combination of downstream signaling activation states to determine the selective gene induction and cell fate under viral infection.

    (A) Immunoblot analyses of the extracts from A549 RIG-IWT or RIG-I6KR cells after SeV infection for 0, 6, 12, and 24 hours with indicated antibodies. (B) Quantification of immunoblots in (A). (C and D) (Left) Flow cytometric analyses of RIG-IWT and RIG-I6KR A549 cells infected with VSV-eGFP (MOI, 0.1 or 1) for indicated time points. Cells were treated with propidium iodide (PI) for 15 min before flow cytometric analyses. (Right) Cell mortality after infection for 24 hours in the left panel. (E) Illustration of specific gene transcription regulated by multisite ubiquitination of RIG-I in antiviral signaling. *P < 0.05, **P < 0.01, and ***P < 0.001 versus RIG-IWT cells.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1701764/DC1

    fig. S1. Generation of RIG-I KO and its mutant-rescued cell lines.

    fig. S2. RIG-I8KR failed to induce ISG expression as well as the antiviral capability.

    fig. S3. Ubiquitination and functional assays support the stratified mechanism of RIG-I–N multisite ubiquitination.

    fig. S4. Antiviral capability of various RIG-I mutants.

    fig. S5. K172R mutation of RIG-I does not influence conjugated ubiquitination of RIG-I but strongly impairs RIG-I unanchored ubiquitination.

    fig. S6. The expression patterns of ISGs induced by RIG-IWT, RIG-IK164R, and RIG-IK172R do not show obvious difference.

    fig. S7. Illustration of RIG-I oligomerization detection system.

    fig. S8. Steady state of poly(I:C)-induced type I IFN activation in RIG-IWT 293T cells.

    fig. S9. Mathematical analysis data fit experimental results well.

    fig. S10. Dynamics of the protein levels of E3 ligases show subtle difference on the ultrasensitivity of RIG-I activation.

    fig. S11. Transcriptome analysis of RIG-IWT, RIG-IK164R, RIG-IK172R, and RIG-I6KR reconstructed cells.

    fig. S12. Dendrograms for randomness in gene expression under RIG-IWT and RIG-I6KR conditions.

    fig. S13. ISG inductions in each cluster.

    fig. S14. Function of E3 ligases in RIG-I–induced type I IFN activation and the knockdown efficiency of indicated E3 ligase’s siRNAs.

    fig. S15. Steady state of RIG-I–induced type I IFN activation in WT 293T cells.

    fig. S16. Activation slope of transcription factors and transcription factor kinases during SeV infection.

    table S1. Parameter values in the model.

    table S2. The number of equations and different ubiquitinated RIG-I that can form tetramers under different conditions.

    Supplementary Methods

    Excel 1. Top 2000 log2FC values.

    Excel 2. Top 2000 FPKM values.

    References (4048)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Generation of RIG-I KO and its mutant-rescued cell lines.
    • fig. S2. RIG-I8KR failed to induce ISG expression as well as the antiviral capability.
    • fig. S3. Ubiquitination and functional assays support the stratified mechanism of RIG-I–N multisite ubiquitination.
    • fig. S4. Antiviral capability of various RIG-I mutants.
    • fig. S5. K172R mutation of RIG-I does not influence conjugated ubiquitination of RIG-I but strongly impairs RIG-I unanchored ubiquitination.
    • fig. S6. The expression patterns of ISGs induced by RIG-IWT, RIG-IK164R, and RIG-IK172R do not show obvious difference.
    • fig. S7. Illustration of RIG-I oligomerization detection system.
    • fig. S8. Steady state of poly(I:C)-induced type I IFN activation in RIG-IWT 293T cells.
    • fig. S9. Mathematical analysis data fit experimental results well.
    • fig. S10. Dynamics of the protein levels of E3 ligases show subtle difference on the ultrasensitivity of RIG-I activation.
    • fig. S11. Transcriptome analysis of RIG-IWT, RIG-IK164R, RIG-IK172R, and RIG-I6KR reconstructed cells.
    • fig. S12. Dendrograms for randomness in gene expression under RIG-IWT and RIG-I6KR conditions.
    • fig. S13. ISG inductions in each cluster.
    • fig. S14. Function of E3 ligases in RIG-I–induced type I IFN activation and the knockdown efficiency of indicated E3 ligase’s siRNAs.
    • fig. S15. Steady state of RIG-I–induced type I IFN activation in WT 293T cells.
    • fig. S16. Activation slope of transcription factors and transcription factor kinases during SeV infection.
    • table S1. Parameter values in the model.
    • table S2. The number of equations and different ubiquitinated RIG-I that can form tetramers under different conditions.
    • Supplementary Methods
    • References (40–48)

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    Other Supplementary Material for this manuscript includes the following:

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