Research ArticleCELL DEATH

OAS1/RNase L executes RIG-I ligand–dependent tumor cell apoptosis

See allHide authors and affiliations

Science Immunology  16 Jul 2021:
Vol. 6, Issue 61, eabe2550
DOI: 10.1126/sciimmunol.abe2550
  • Fig. 1 RIG-I ligands execute tumor cell death by an RIG-I–independent, IFN-I–primed mechanism.

    (A) 1205Lu human melanoma cells of the indicated genotype were stimulated with 80 nM 3p-RNA or nonstimulatory control RNA (CTRL) and analyzed for cell death by annexin A5 assay and for MHC-I expression after 48 hours (n = 2). MFI, median fluorescence intensity. (B) ELISA for human IFN-β in the cell supernatant after stimulation with 160 nM 3p-RNA or controls for 48 hours (n = 3). (C) Schematic of coculture assay and gating strategy for flow cytometric analysis. (D) WT and RIG-I–deficient 1205Lu cells were cultured alone or cocultured as depicted in (C), treated with 160 nM 3p-RNA or CTRL, and analyzed by flow cytometry (n = 3, Student’s t test). (E) 1205Lu cells of indicated genotype were prestimulated with IFN-α (1000 U/ml) 18 hours before transfection with 40, 80, or 160 nM 3p-RNA or CTRL. Viability was assessed by CTB assay 48 hours after RNA transfection (n = 3, Student’s t test). (F) WT and RIG-I–deficient or WT and RIG-I/IFNAR1–deficient 1205Lu cells were cultured alone or cocultured, treated with 160 nM 3p-RNA or CTRL, and analyzed by flow cytometry (n = 3, one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test; asterisks indicate results of post hoc test.) (G) WT and IRF3−/− 1205Lu cells were prestimulated with IFN-α (1000 U/ml) or mock for 18 hours before transfection of indicated RNAs as shown (160 nM). Viability was measured by CTB assay (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). (H) Viability of WT and IFNAR1−/− 1205Lu cells 48 hours after transfection with 3p-RNA or CTRL (160 nM) (n = 3, Student’s t test). (I) CTB assay of WT and DDX58−/− cells transfected with total RNA isolated from WT or M51R VSV-infected cells (1 μg/ml) for 48 hours. DDX58−/− cells were treated with IFN-α or mock (ø) for 18 hours before RNA transfection [n = 2, one-way ANOVA (WT) or two-way ANOVA (DDX58−/−), followed by Sidak’s multiple comparisons test]. Asterisks indicate results of post hoc test. Asterisks on bars represent comparison to CTRL-RNA. Means + SEM of independent experiments are shown; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

  • Fig. 2 3p-RNA specifically binds OAS1 and activates RNase L.

    (A) Volcano plots of differential gene expression (log2 FC) for indicated conditions upon 3p-RNA treatment. (B) Top 30 induced genes (log2 FC) annotated with the GO:MF term double-stranded RNA binding (blue) or RNA binding (gray) as determined by RNA-seq in 1205Lu DDX58−/− cells stimulated for 4 hours with IFN-α compared with untreated control. (C) IFN-α–primed 1205Lu DDX58−/− cells were transfected with FastAP- or mock-treated 3p-RNA as indicated. Cell death induction was measured 48 hours after transfection (n = 3, Student’s t test). (D) Volcano plot of relative LFQ of proteins bound to 3p-RNA-biot compared with FastAP-treated 3p-RNA-biot (OH-RNA-biot) as quantified by LC-MS/MS (n = 5). Proteins up-regulated in (A) with GO:MF term double-stranded RNA binding are highlighted in blue, those significantly enriched are indicated in green. (E) Western blot validation of MS results. 3p-RNA-biot or OH-RNA-biot was bound to streptavidin beads and used for affinity purification (AP) of proteins in lysates from unstimulated or IFN-α–stimulated (18 hours) cells. Thirty micrograms of protein lysate was used as input. Representative of three independent experiments. (F) OAS1 activation by chemically synthesized RNAs as measured by pyrophosphate production using a chromogenic assay. CTRL, non-immunostimulatory control RNA; 3p-RNA/OH-RNA, custom-synthesized RNAs with (3p) or without (OH) 5′-triphosphate group; 3′-ssPy, dsRNA with single-stranded 3′-pyrimidine overhang (mean ± SD of n = 3, one-way ANOVA, followed by Dunnett’s multiple comparisons test; asterisks indicate results of post hoc test). (G and H) rRNA integrity analysis of total RNA isolated from 1205Lu DDX58−/− cells using RNA BioAnalyzer. (G) IFN-α–primed cells were transfected with OH-RNA, 3p-RNA (both 80 nM) or poly(I:C) (1 μg/ml) for 6 hours. Left: Representative image of four independent experiments. Right: Quantification of RNA degradation by RNA-integrity number (RIN) (n = 4, Student’s t test). (H) Cells untreated or treated with IFN-α (18 hours), 3p-RNA (6 hours), or IFN-α and then 3p-RNA. Representative of at least three independent experiments. Unless stated otherwise, means + SEM of independent experiments are shown; *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 3 The OAS/RNase L system mediates the effector phase of apoptosis induction by 3p-RNA.

    (A) rRNA integrity analysis of total RNA isolated from 1205Lu cells with indicated genotypes untreated or treated with IFN-α (1000 U/ml, 18 hours), transfected with 3p-RNA (12 hours, 160 nM), or treated with IFN-α (18 hours), and then with 3p-RNA (12 hours). Representative of two independent experiments. (B) Cell death induction in 1205Lu WT and RNASEL−/− cells treated with increasing doses of 3p-RNA (40, 80, and 160 nM) for 48 hours (n = 4, Student’s t test). (C) Kinetic analysis of cell death induction using xCELLigence real-time cell analyzer. WT and indicated KOs in 1205Lu cells were plated on an E-Plate 96 PET with or without IFN-α (1000 U/ml) for 16 hours and then transfected with 3p-RNA (160 nM). Impedance was analyzed for 48 hours after 3p-RNA transfection. Mean of n = 3. (D) Western blot analysis of 1205Lu DDX58−/− cells overexpressing OAS1, OAS2, or OAS3 or empty vector control with or without 18-hour stimulation with Dox (1 μg/ml) or IFN-α (1000 U/ml). Representative of two independent experiments. Relative transgene expression to IFN-α–stimulated sample after normalization to loading control is shown below. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) CTB assay of cells as in (D) with or without stimulation with either IFN-α (1000 U/ml) or Dox (0.01, 0.1, and 1 μg/ml) and treatment with 160 nM 3p-RNA for 48 hours (n = 5, Student’s t test). (F) Viability of 1205Lu WT and OAS1−/− cells 48 hours after RNA transfection with 160 nM, normalized to untreated (n = 3, Student’s t test). (G) CTB assay of 1205Lu WT and RNASEL−/− cells treated with total RNA (1 μg/ml) from cells infected with indicated VSV or SeV for 48 hours [n = 7 (CTRL-RNA), 5 (VSV-M51R), and 4 (VSV-WT and SeV), Student’s t test]. (H) Flow cytometric analysis of viability of WT or RNase L–deficient B16F10 cells 48 hours after transfection (n = 3, Student’s t test). RNA preparations as described in (G) were used. Unless stated otherwise, means + SEM of independent experiments are shown; *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 3p-RNA induces intrinsic apoptosis.

    (A) 1205Lu cells of indicated genotypes were transfected with 3p-RNA with or without prestimulation with IFN-α (1000 U/ml) for 18 hours. Caspase-positive, fixable viability dye (FVD)–negative cells were measured using Live Cell Caspase Probe via flow cytometry 30 hours after transfection (n = 3, Student’s t test). (B) 1205Lu cells were incubated with the pancaspase inhibitor z-VAD (20 μM, + or 80 μM, ++) or solvent control for 1 hour before RNA transfection, and cell death induction was measured after 48 hours (n = 2). (C) Western Blots of BAX and BAK expression in WT and RIG-I–deficient cells treated with sgRNAs targeting BAX and BAK1 (sgBAX/BAK) or an irrelevant control (sgCTRL). Representative of two independent experiments. (D) Cell death induction by 3p-RNA treatment (160 nM) 48 hours after transfection in IFN-α–primed DDX58−/− or WT 1205Lu cells (n = 2, Student’s t test). Means + SEM of independent experiments are shown; *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 RNase L–dependent translational arrest facilitates NOXA-mediated MCL1 depletion.

    (A) Measurement of puromycin (Puro) incorporation detected by anti–puromycin (Puro) blot (top image) and total protein (Stain-Free technology, bottom image) in IFN-α–primed 1205Lu WT or RNASEL−/− cells treated with CTRL, 3p-RNA (both 160 nM), or CHX (10 μg/ml) for 6 hours. Representative of two independent experiments. (B) Cell death induction in 1205Lu WT cells 24 hours after stimulation with small-molecule inhibitors of BCL2 family proteins (all used at 1 μM) in the presence or absence of CHX (1 μg/ml) [n = 2 to 4, two-way ANOVA, followed by Tukey’s multiple comparisons test; asterisks indicate results of post hoc test, and asterisks above bars indicate comparison to dimethyl sulfoxide (DMSO)–treated sample]. (C) Immunoblot of NOXA expression in 1205Lu WT cells treated with an sgRNA targeting the NOXA gene PMAIP1 (sgNOXA) or nontargeting control (sgCTRL). Representative of two independent experiments. (D) Cell death induction in the cell lines described in (C) 18 hours after 3p-RNA transfection (n = 3, Student’s t test). (E) NOXA expression assessed by Immunoblot in 1205Lu WT cells treated with 3p-RNA or poly(I:C) for 6 hours. Representative of two independent experiments. (F) Cell death induction by poly(I:C) (1 μg/ml) or 3p-RNA (160 nM) with or without CHX (10 μg/ml) and with or without transfection reagent (n = 2 to 4, one-way ANOVA, followed by Sidak’s multiple comparisons test). Uncomplexed poly(I:C) was added 6 hours before treatment with CHX. CHX, transfected poly(I:C), or transfected 3p-RNA was added for 12 hours before analysis. (G) qRT-PCR analysis of ACTB (encoding for β-actin), MCL1, and PMAIP1 (encoding for NOXA) expression in WT and RNase L–deficient 1205Lu cells at indicated time points after 3p-RNA treatment (bars represent mean differences in crossing point (Cp) value between mock and 3p-RNA–treated cells ± SEM of three to four independent experiments, Student’s t test; asterisks above bars indicate comparison to untreated control). (H) Immunoblots of IFN-α–primed 1205Lu WT and RNASEL−/− cells stimulated with 3p-RNA for indicated time points or with CHX (10 μg/ml) for 14 hours. Immunoblot shows representative results of three independent experiments. (I) Immunoblot for MCL-1 and NOXA expression of IFN-α–primed 1205Lu WT cells treated with an sgRNA targeting PMAIP1 (sgNOXA) or nontargeting control (sgCTRL) stimulated with 3p-RNA for indicated time points or with CHX (10 μg/ml) for 14 hours (sgCTRL only). Representative results of two independent experiments. (J) Schematic representation of the proposed mechanism of MCL-1 depletion in response to RNase L activation. Unless otherwise stated, means + SEM of independent experiments are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • The PDF file includes:

    • Figs. S1 to S5
    • Table S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Immunology

Navigate This Article