RNA-binding proteins hnRNPM and ELAVL1 promote type-I interferon induction downstream of the nucleic acid sensors cGAS and RIG-I

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  Figure 1. hnRNPM promotes type I IFN induction downstream of both cGAS and RIG-I. (A) Fluorescence microscopy of HEK293FT cells transiently transfected with hnRNPM-GFP (top) or GFP (bottom) together with a control vector or vectors encoding CVB3 3 C protease-FLAG- or PV 3C protease-FLAG (CMV or EF1α promotor). (B) Expression of HNRNPM mRNA in THP-1 WT and cells expressing control shRNA (shC001) or hnRNPM-specific shRNAs (shhnRNPM.1, shhnRNPM.2) (from left to right: n = 5, 5, 5, 5 independent experiments). (C) MTT assay of the cells depicted in (B) (from left to right: n = 3, 3, 3, 3 independent experiments). (D) ISRE reporter activation in the cells depicted in (B) 20 h after stimulation of RIG-I with 5′ppp-dsRNA (0.1 µg/ml), of cGAS with pDNA (0.1 µg/ml) or G3-YSD (0.5 µg/ml), of TLR8 with TL8-506 (1.0 µg/ml), of TLR1/2 with Pam3CSK4 (0.5 µg/ml), or of STING with 2'3′-cGAMP (10 µg/ml). ctrl, non-stimulated (stimuli from left to right: n = 14, 14, 14, 14, 7, 3, 13 independent experiments). (E) ISRE reporter activation in the cells depicted in (B) 20 h after stimulation of IFNAR with IFNα (1000 U/ml). ctrl, non-stimulated (stimuli from left to right: n = 4, 4 independent experiments). (F)…

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  Figure EV1. hnRNPM functions upstream of IFNAR. (A) Expression of IFNB1 mRNA in THP-1 cells expressing control shRNA (shC001) or hnRNPM-specific shRNAs (shhnRNPM.1, shhnRNPM.2) 6 h after stimulation of RIG-I with 5′ppp-dsRNA (0.1 µg/ml), of cGAS with pDNA (0.1 µg/ml) or G3-YSD (0.5 µg/ml), of TLR8 with TL8-506 (1.0 µg/ml), of TLR1/2 with Pam3CSK4 (0.5 µg/ml), of STING with 2'3′-cGAMP (10 µg/ml), or of IFNAR with IFNα (1000 U/ml). ctrl, non-stimulated (mean ± SD; stimuli from left to right: n = 3, 3, 2, 2, 2, 2, 2, 2 independent experiments). (B) Expression of CXCL10 mRNA in the cells depicted in (A) 6 h after stimulation with the indicated stimuli (mean ± SD; stimuli from left to right: n = 3, 3, 3, 3, 3, 3, 3, 3 independent experiments). (C) Quantification of pIRF3-Ser396 levels shown in Fig. 1I (mean ± SD; stimuli from left to right: n = 2, 2, 2 independent experiments). (D) Immunoblot analysis of pSTAT1-Tyr701 induction in THP-1 WT and cells expressing control shRNA (shC001) or hnRNPM-specific shRNAs (shhnRNPM.1, shhnRNPM.2) after stimulation with IFNα (1000 U/ml). ctrl, non-stimulated. One representative experiment of two independent experiments is shown. (E) Immunoblot analysis of THP-1…

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  Figure 2. Interactome of hnRNPM. (A) Schematic workflow of the combined AP-MS-RNAi screening approach to identify immune-relevant interactors of hnRNPM. (B) hnRNPM-GFP and GFP were immunoprecipitated from lysates of non-stimulated THP-1 cells and bound proteins were identified by LC-MS/MS. The Volcano plot shows the log2 fold change enrichment of proteins detected in the hnRNPM-GFP vs. GFP affinity purification, plotted against the statistical significance (−log10P). Significantly enriched proteins were determined by two-sided Welch’s t-tests (S0 = 0.1, permutation-based FDR < 0.05). Colors indicate pathway annotations and the majority of labeled proteins were functionally evaluated by RNAi.

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  Figure EV2. hnRNPM interacts with proteins connected to immune system processes. hnRNPM-GFP and GFP were immunoprecipitated from lysates of non-stimulated THP-1 cells. Differential interactors of hnRNPM were analyzed by STRING enrichment and annotated with GO terms enriched among hnRNPM interactors. hnRNPM interactors not annotated with these GO terms are shown at the bottom. cluster (C) 1–9. The statistical tests used are described in detail in the methods.

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  Figure 3. ELAVL1 interacts with hnRNPM to regulate type I IFN induction downstream of both cGAS and RIG-I. (A) Left panel: Expression of ELAVL1 mRNA in THP-1 cells expressing control shRNAs (shC001, shC002) or ELAVL1-specific shRNAs (shELAVL1.1, shELAVL1.2) (mean ± SD, one-way ANOVA, Dunnett’s multiple comparisons test; from left to right: n = 3, 3, 3, 3 independent experiments), right panel: ISRE reporter activation 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), or G3-YSD (0.5 µg/ml) (mean ± SD, two-way ANOVA, Dunnett’s multiple comparisons test; stimuli from left to right: n = 3, 3, 3, 3 independent experiments). ctrl, non-stimulated. (B) Expression of HNRNPM and ELAVL1 mRNA in primary human fibroblasts stably expressing control shRNA (shC001) or shRNAs against HNRNPM or ELAVL1 (shRNA.1, shRNA.2) (mean ± SD; from left to right: n = 2, 2, 2, 2, 2, 2 independent experiments). (C) CXCL10 ELISA with supernatants of the cells depicted in (B) 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml). ctrl, non-stimulated (mean ± SD, two-way ANOVA, Dunnett’s multiple comparisons test; stimuli from left to right: n = 3, 3 independent experiments). (D) CXCL10 ELISA with…

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  Figure EV3. Functional RNAi screen of hnRNPM interactors. (A) mRNA expression of the indicated targets (y-axis) in THP-1 cells expressing control shRNAs (shC001, shC002) or target-specific shRNAs (shRNA.1, shRNA.2). Target mRNA expression was normalized to GAPDH mRNA and then normalized to shC001-expressing cells. Data of two or more independent experiments are shown. (B) ISRE reporter activation in the cells depicted in (A) 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml) or pDNA (0.1 µg/ml). Luciferase signals were normalized to shC001-expressing cells of the respective condition. Data of two or more independent experiments are shown. (C) Nuclear extracts (NE) and cytoplasmic extracts (CE) were prepared from non-stimulated THP-1 cells or from cells stimulated with G3-YSD (0.5 µg/ml) for 3 h and analyzed by immunoblotting with the indicated antibodies. One representative experiment of two independent experiments is shown. (D) NE and CE were prepared from non-stimulated THP-1 cells or from cells stimulated with 5′ppp-dsRNA (0.1 µg/ml) for 3 h and analyzed by immunoblotting with the indicated antibodies. One representative experiment of two independent experiments is shown.

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  Figure 4. ELAVL1 promotes the cGAS- and RIG-I-mediated induction of type I IFNs and activation of NF-κB. (A) Immunoblot analysis of THP-1 WT and ELAVL1 KO cells using an ELAVL1-specific antibody. (B) ISRE reporter activation in the cells depicted in (A) 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), G3-YSD (0.5 µg/ml), or IFNα (1000 U/ml). ctrl, non-stimulated (left panel: stimuli from left to right, n = 4, 4, 4, 4 independent experiments; right panel: stimuli from left to right, n = 3, 3 independent experiments). (C) ISRE reporter activation in THP-1 WT, ELAVL1 KO (#1), and ELAVL1 KO (#1) expressing ELAVL1-FLAG or GFP 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), G3-YSD (0.5 µg/ml), or Pam3CSK4 (0.5 µg/ml). ctrl, non-stimulated (stimuli from left to right: n = 4, 4, 4, 4, 4 independent experiments). (D) ISRE reporter activation in the cells depicted in (C) 20 h after stimulation with 2'3′-cGAMP (10 µg/ml). ctrl, non-stimulated (stimuli from left to right: n = 3, 3 independent experiments). (E) ISRE reporter activation in the cells depicted in (C) 20 h after stimulation with IFNα (1000 U/ml). ctrl, non-stimulated (stimuli from left to right:…

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  Figure EV4. ELAVL1 is a potent positive regulator of cGAS and RIG-I signaling. (A) ISRE reporter activation in THP-1 WT, ELAVL1 KO (#1), ELAVL1 KO (#1) expressing ELAVL1-FLAG or GFP, RIG-I KO (clones #1–2), MAVS KO (clones #1–2), and cGAS KO cells 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), G3-YSD (0.5 µg/ml), Pam3CSK4 (0.5 µg/ml), or IFNα (5000 U/ml) (mean ± SD; stimuli from left to right: n = 3, 3, 3, 3, 3, 3 independent experiments). ctrl, non-stimulated. (B) ISRE reporter activation in the cells depicted in (A) 20 h after challenge with 2'3′-cGAMP (10 µg/ml) (mean ± SD; stimuli from left to right: n = 3, 3 independent experiments). ctrl, non-stimulated. (C) NF-κB reporter activation in the cells depicted in (A) 20 h after challenge with the indicated stimuli (mean ± SD; stimuli from left to right: n = 3, 3, 3, 3, 3, 3, 3 independent experiments). (D) MTT assay of THP-1 WT, ELAVL1 KO (#1), and ELAVL1 KO (#1) expressing ELAVL1-FLAG or GFP 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), G3-YSD (0.5 µg/ml), 2'3′-cGAMP (10 µg/ml), Pam3CSK4 (0.5 µg/ml), or IFNα (1000 U/ml). ctrl, non-stimulated (stimuli from left to right: n = 11, 6, 6, 6, 3,…

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  Figure EV5. ELAVL1 regulates signal transduction downstream of cGAS and RIG-I. (A) ISRE reporter activation in THP-1 WT, IFNAR2 KO and IFNAR2/ELAVL1 double-KO cells (clones #1–2, ELAVL1 gRNA AN) 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml), pDNA (0.1 µg/ml), G3-YSD (0.5 µg/ml), Pam3CSK4 (0.5 µg/ml), or IFNα (5000 U/ml) (mean ± SD: stimuli from left to right: n = 2, 2, 2, 2, 2, 2 independent experiments). ctrl, non-stimulated. (B) ISRE reporter activation in shC001-expressing THP-1 cells and IFNAR2 KO cells expressing shC001, shhnRNPM.1, or shhnRNPM.2 20 h after stimulation with 5′ppp-dsRNA (0.1 µg/ml) or pDNA (0.1 µg/ml) (mean ± SD; stimuli from left to right: n = 10, 9, 9 independent experiments).). ctrl, non-stimulated. (C) 3′-mRNA sequencing of total RNA from IFNα-stimulated (1000 U/ml, 6 h) THP-1 ELAVL1 KO (#1) and WT cells. Wald test was used to identify differentially expressed genes, with P values adjusted for multiple testing using the Benjamini–Hochberg procedure. The Volcano plot correlates the gene expression (log2 fold change of ELAVL1 KO vs. WT cells) with the -log10 adjusted P value (Padjusted). Significantly regulated genes were defined as Padjusted < 0.05 and log2 fold…

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  Figure 5. hnRNPM and ELAVL1 interact with TBK1, IKKε, IKKβ, and NF-κB p65. (A) GFP-specific beads were used to immunoprecipitate hnRNPM-GFP and GFP from lysates of non-stimulated or pDNA-stimulated (0.1 µg/ml, 3 h) THP-1 cells. If indicated, IPs were treated with RNase A (100 µg/ml, 1.5 h). The IPs and 5.0% of the cleared cellular lysate used for IP (input) were analyzed by immunoblotting with the indicated antibodies. IPs and input controls of hnRNPM-GFP and GFP were analyzed on the same membrane, with empty lanes removed. (B) FLAG tag-specific beads were incubated with lysates of WT or ELAVL1-FLAG-expressing HEK293FT cells. If indicated, the IPs were treated with RNase A (100 µg/ml, 1.5 h). IPs and input controls were analyzed by immunoblotting with the indicated antibodies. (C) Differentiated THP-1 cells stably expressing hnRNPM-GFP or GFP were transfected with Alexa Fluor 647-labelled G3-YSD (0.5 µg/ml, 4 h) or left non-stimulated (ctrl). Anti-GFP and anti-pTBK1-Ser172 monoclonal antibodies were used to analyze the interactions between hnRNPM-GFP/GFP and pTBK1-Ser172 in cellulo by PLA. Z stack images were recorded on a confocal microscope. Image sequences of hnRNPM-GFP-expressing cells…