Interleukin-2-mediated NF-κB-dependent mRNA splicing modulates interferon gamma protein production

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  Figure 1. IL-2 induces post-transcriptional stability of IFNG mRNA. Healthy human CD56 + NK cells were treated with IL-2 (100 U/mL), IL-12 (10 ng/mL) or both for 6 h. Supernatants and cell lysates were collected for (A) IFNγ protein quantification via ELISA (p = 0.037 for both IL-2 versus IL-12 + IL-2 and IL-12 versus IL-12 + IL-2) or (B) qPCR analysis of IFNG induction respectively (IL-12 versus IL-12 + IL-2 p = 0.01). IFNG expression normalized to HPRT1. NK92 cells were stimulated 3 or 6 h as in (A) and supernatants and cell lysates collected for (C) ELISA (IL-12 versus IL-12 + IL-2, 6 h stimulation, p = 0.0009) and (D) qPCR (IL-12 versus IL-12 + IL-2, 6 h stimulation, 0.0047) respectively, IFNG induction normalized to HPRT1. (E) qPCR analysis of total versus 4SU labeled IFNG for IL-12 versus IL-12 + IL-12 stimulated cells, normalized to HPRT1 expression. (F) Time course of IFNG mRNA stability in absence of nascent transcription during IL-2 stimulation, normalized to IFNG levels at 3 h IL-12 treatment before addition of ActD (IL-12 versus IL-12 + IL-2, 2 h treatment, p = 0.0097). Data information: Data in (A–E) are mean ± SEM of 3 biological replicates, (A), (B) analyzed by one-way ANOVA…

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  Figure EV1. IL-2 stimulation does not globally affect transcript stability or protein translation in NK92 cells. (A) Schematic of IFNγ bioassay workflow for quantification of IFNγ in supernatants of treated NK92 cells via Huh7 Gaussia luciferase IRF1 GAS(5x) reporter cells. (B) Bioassay quantification of IFNγ protein production using supernatants from NK cells stimulated 24 h with IL-2 (100 u/mL), IL-12 (10 ng/mL) or IL-12 + IL-2 (IL-12 versus IL-12 + IL-2 stimulation, p = 0.0037). (C) qPCR analysis of 4SU labeled IFNG transcripts compared with total (4SU labeled + unlabeled) IFNG induction during IL-12 versus IL-12 + IL-2 treatment; normalized to HPRT1. Time course of (D) RELC, (E) HNRNPU, and (F) GAPDH mRNA stability in absence of nascent transcription during IL-2 stimulation, normalized to RELC, HNRNPU, and GAPDH levels at 3 h IL-12 treatment before addition of ActD, respectively. (G) qPCR analysis of IFNG mRNA induction during IL-2 stimulation in presence and absence of nascent translation, using cycloheximide (CHX) treatment (100 μg/mL) to halt protein synthesis. Normalized to HPRT1 expression (H) Immunoblot depicting puromycin incorporation for cycloheximide protein synthesis halt…

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  Figure 2. IL-2 promotes IFNG mRNA splicing. (A) Primer design for PCR amplification of intra-exonic regions of IFNG mRNA; primers used in (B), (Fig. EV1B). (B) Bioanalyzer analysis of PCR amplification of intra-exonal regions of IFNG isolated from fractionated nuclei from NK92 cells stimulated with IL-12 or IL-12 + IL-2 for 6 h. (C) Schematic of SYBR probe design for qPCR amplification of intronic regions of IFNG in (D–F), and all further experiments quantifying intron retention (D) SYBR qPCR analysis of IFNG intron retention in nuclear lysates during stimulation with IL-12 or IL-12 + IL-2 for 3 or 6 h. Intron expression normalized to IFNG 5’UTR, representing total mature plus unspliced IFNG mRNA as quantified by amplification of the region spanning the 5’UTR into the coding region of Exon 1, depicted in (C) as control (Intron 1: 3 h, IL-12 versus IL-12 + IL-2, p = 0.049, 6 h, p = 0.0006; Intron 2: 6 h, p = 0.002; Intron 3: 6 h, p = 0.014). (E) SYBR qPCR analysis of IFNG intron retention in NK92 whole cell lysates during a time course of IL-12 or IL-12 + IL-2 stimulation for 7.5 h, normalized to the IL-12 stimulation condition and to the IFNG 5’UTR within each timepoint (IL-12 versus IL-12…

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  Figure EV2. IL-2 mediates IFNG intronic splicing during nascent translation and upon cycloheximide treatment. (A) Schematic of NK92 stimulation and processing for determining IFNG intron retention via PCR as seen in Fig. 2B, EV1B. (B) Image of gels depicting PCR products from amplification of intra-exonal regions of IFNG from nuclear and cytosol fractionated NK92 cells stimulated with IL-12 or IL-12 + IL-2 for 3 or 6 h. Blue arrows depict fully spliced products while red arrows depict amplified intronic regions. (C) total spliced IFNG induction (IL-12 versus IL-12 + IL-2 stimulation, 3 h treatment p = 0.04; 6 h, p = 0.044) normalized to HPRT1 expression during stimulations outlined in Fig. 2D. (D) SYBR qPCR analysis of IFNG intron retention in whole cell lysates during stimulation with IL-12 for 3 h with or without subsequent 3 h IL-2 stimulation. Each stimulation condition normalized to mock treatment condition. Intron expression normalized to IFNG 5’UTR, representing total mature plus unspliced IFNG mRNA as quantified by amplification of the region spanning the 5’UTR into the coding region of Exon 1 as control (Intron 1, IL-12 versus IL-12 + IL-2 stimulation, p = 0.014; Intron 2, p =…

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  Figure EV3. ERK signaling does not post-transcriptionally regulate IFNG mRNA processing. (A) Schematic of canonical IL-2R signaling. (B) Immunoblot confirming effect of inhibitor PD98059 (10 μM) on preventing phosphorylation ERK1/2 downstream of MEK1/2 inhibition during IL-12 and IL-2 treatment in NK92 cells (C) schematic of PD98059 treatment in determining effect on IFNγ induction and splicing. (D) Gaussia luciferase bioassay for IFNγ protein quantification (E) total IFNG transcript induction and (F) IFNG intron retention upon treating cells with PD98059 (25 μM) prior to IL-2 treatment, normalized to total spliced plus unspliced IFNG (Mock treatment, IL-12 versus IL-12 p < 0.0001; PD98059 treatment, IL-12 versus IL-12 + IL-2 p = 0.017). Data information: Data in (D–F) is mean ± SEM for 3 or 4 biological replicates. (B) is representative of 3 biological replicates. (D–E) analyzed two-way ANOVA with Turkey’s test for multiple comparisons and (F) by paired T test with Holm-Šídák method for multiple corrections. *p ≤ 0.05, ****p ≤ 0.0001, n.s. is not significant.

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  Figure 3. Post-transcriptional IL-2 mediated splicing of IFNG is NF-κB-dependent. (A) Schematic for inhibition of NF-κB signaling with BAY-11 during IL-2 stimulation and Gaussia luciferase bioassay for IFNγ protein quantification (IL-12 comparison between treatments: p = 0.009; IL-12 + IL-2 comparison: p = 0.0006). (B) qPCR analysis of total IFNG mRNA induction normalized to HPRT1 (Mock versus BAY-11 treatment during IL-12 + IL-2 treatment, p = 0.023) and (C) SYBR qPCR analysis of IFNG mRNA intron retention upon inhibiting NF-κB signaling with BAY-11 (10 μM) treatment prior to IL-2 treatment (Intron 1: Mock treatment, IL-12 versus IL-12 + IL-2, p = 0.005; Intron 2: Mock treatment, IL-12 versus IL-12 + IL-2, p = 0.001). (D) mature IFNG mRNA induction (Mock versus ActD+BAY-11 conditions during IL-12 + IL-2 treatment, p = 0.007) and (E) IFNG intron retention upon inhibition of both nascent transcription and NF-κB signaling prior to IL-2 treatment; normalized to HPRT1 and to 5’UTR of IFNG mRNA, respectively (Intron 1: Mock treatment, IL-12 versus IL-12 + IL-2, p = 0.048; Intron 2: Mock treatment, p = 0.016). (F) IFNG mRNA induction normalized to HPRT1 and to the no stimulation condition within…

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  Figure EV4. NF-κB signaling downstream of IL-2 is required for post-transcriptional regulation of IFNG mRNA. (A) Schematic depicting alternative signaling through Akt downstream of the IL-2 receptor in NK cells (B) immunoblot for total and phosphorylated NF-κB p65 NK cells treated with IL-12 or IL-12 + IL-2 for 3 or 6 h with GAPDH as loading control (C) qPCR analysis of IFNG induction upon pre-treatment with the NF-κB inhibitor BAY-11, prior to any stimulation (Mock treatment, IL-12 versus IL-12 + IL-2 comparison p < 0.0001). (D) IFNG mRNA induction normalized to HPRT1 with all samples normalized to the no stimulation condition within the mock treatment to confirm functioning ActD halt of nascent transcription (Mock treatment, IL-12 versus IL-12 + IL-2, p = 0.0397; ActD treatment, IL-12 versus IL-12 + IL-2, p = 0.0257). (E) qPCR analysis of total IFNG mRNA induction (Mock versus MLN4924 treatment during IL-12 + IL-2 stimulation, p = 0.015) and (F) SYBR qPCR analysis of IFNG mRNA intron retention upon inhibiting NF-κB signaling with MLN4924 (10 μM), (Intron 1, Mock treatment, IL-12 versus IL-12 + IL-2 p = 0.049; Intron 2 Mock treatment p = 0.0004). Data information: (B) is representative of 5…

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  Figure 4. NF-κB signaling is required for PMA-mediated splicing of IFNG. (A) Bioassay quantification of IFNγ protein production using Huh7 Gaussia luciferase IRF1 GAS(5x) reporter cells treated for 24 h with supernatants collected after 24 h NK92 stimulation with IL-2 (100 U/mL), IL-12 (10 ng/mL), PMA (10 nM), or a combination (IL-12 versus IL-12 + IL-2 and PMA versus PMA + IL-2, both p < 0.0001). Bioassay quantification of IFNγ protein production and (C) qPCR analysis of IFNG induction during stimulation with PMA (10 nM), Ionomycin (1 μg/mL), IL-12 (10 ng/mL) or a combination for 24 h (B), PMA versus PMA + IL-12, p < 0.0001; Iono+ IL-12 versus PMA + IL-12, p < 0.0001) or 6 h (C), PMA versus IL-12 + PMA, p < 0.0001; Iono+IL-12 versus PMA + IL-12, p < 0.0001). (D) SYBR qPCR analysis of IFNG mRNA intron retention of total lysates from cells treated with IL-12 (10 ng/mL), IL-12 + PMA (10 nM), or IL-12+Ionomycin (1 μg/mL) for 6 h, normalized to total mature plus unspliced IFNG (Intron 1, IL-12 versus IL-12 + PMA, p = 0.0034; Intron 2, IL-12 versus IL-12 + PMA, p = 0.0016). (E) Gaussia luciferase bioassay for IFNγ protein quantification (Mock versus BAY-11 treatment during IL-12 stimulation, p…