Figure 4. The IL-1β – p38α axis promotes...

$The IL-1β–p38α axis promotes mRNA nuclear export and protein production of ILC3 cytokines by disrupting the eIF6/mRNA paraspeckle. (A–C) The biotinylated Il22 CDS mRNAs with or without 3′UTRs were incubated with cell lysates from ILC3s treated with or without IL-1β, and then mRNA binding proteins were isolated and analyzed by MS. (A) The diagram of strategy for screening of IL-22 mRNA 3′UTR-interactome. (B) The mRNA-interactome identified by MS. (C) The Venn diagram shows the interactive analysis with the unique 3′UTR-interactome and TurboID-p38α-interactome. (D) Western blot analysis of the co-IP between p-p38α and eIF6 in MNK-3 cells treated with or without IL-1β. (E) The binding affinity between p38α and eIF6 was measured by Biacore SPR assay. eIF6 was attached to the CM-5 chips and the indicated flow phases were delivered to the Biscore system. (F) Representative images of immunofluorescent staining of eIF6 (red) and smFISH of Il22 mRNA (cyan) in MNK-3 cells. Line traces (white arrow in merged inset) of fluorescence intensity from images. Scale bars, 5 μm. (G) Representative immunofluorescence images of eIF6 (red) in MNK-3 cells after 2 h of IL-1β treatment with or without p38α inhibitor SB203580. The MFI of eIF6 puncta was analyzed by ImageJ. Scale bars, 5 μm. (H) Quantification of eIF6 protein in nuclear and cytosol of MNK-3 cells with or without IL-1β and SB203580 treatment. (I) eIF6-overexpressing MNK-3 cells were constructed via infection with eIF6-expressing retrovirus. IL-22 and GM-CSF production were examined by flow cytometry (top) and ELISA (bottom). (J) eIF6-knock-down MNK-3 cells were constructed via infection with retrovirus containing shRNA against eIF6. IL-22 and GM-CSF production were examined by flow cytometry. (K) eIF6 was knocked-down through retrovirus containing shRNA against eIF6 in p38α-deficient ILC3s. The protein expression of IL-22 and GM-CSF were examined by flow cytometry. (L) Control and eIF6-overexpressing MNK-3 cells were stimulated with IL-1β, IL-23 and PMA/ionomycin. The nuclear and cytosol mRNA were fractionated and analyzed by qPCR. Data are representative of one of two independent experiments (D–L). Each dot (I and K) represents one biological replicate. Dissociation-one phase decay is applied to fit KD (E). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (G and I–L), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F4.$

Figure 4.

The IL-1β – p38α axis promotes mRNA nuclear export and protein production of ILC3 cytokines by disrupting the eIF6/mRNA paraspeckle. (A–C) The biotinylated Il22 CDS mRNAs with or without 3′UTRs were incubated with cell lysates from ILC3s treated with or without IL-1β, and then mRNA binding proteins were isolated and analyzed by MS. (A) The diagram of strategy for screening of IL-22 mRNA 3′UTR-interactome. (B) The mRNA-interactome identified by MS. (C) The Venn diagram shows the interactive analysis with the unique 3′UTR-interactome and TurboID-p38α-interactome. (D) Western blot analysis of the co-IP between p-p38α and eIF6 in MNK-3 cells treated with or without IL-1β. (E) The binding affinity between p38α and eIF6 was measured by Biacore SPR assay. eIF6 was attached to the CM-5 chips and the indicated flow phases were delivered to the Biscore system. (F) Representative images of immunofluorescent staining of eIF6 (red) and smFISH of Il22 mRNA (cyan) in MNK-3 cells. Line traces (white arrow in merged inset) of fluorescence intensity from images. Scale bars, 5 μm. (G) Representative immunofluorescence images of eIF6 (red) in MNK-3 cells after 2 h of IL-1β treatment with or without p38α inhibitor SB203580. The MFI of eIF6 puncta was analyzed by ImageJ. Scale bars, 5 μm. (H) Quantification of eIF6 protein in nuclear and cytosol of MNK-3 cells with or without IL-1β and SB203580 treatment. (I) eIF6-overexpressing MNK-3 cells were constructed via infection with eIF6-expressing retrovirus. IL-22 and GM-CSF production were examined by flow cytometry (top) and ELISA (bottom). (J) eIF6-knock-down MNK-3 cells were constructed via infection with retrovirus containing shRNA against eIF6. IL-22 and GM-CSF production were examined by flow cytometry. (K) eIF6 was knocked-down through retrovirus containing shRNA against eIF6 in p38α-deficient ILC3s. The protein expression of IL-22 and GM-CSF were examined by flow cytometry. (L) Control and eIF6-overexpressing MNK-3 cells were stimulated with IL-1β, IL-23 and PMA/ionomycin. The nuclear and cytosol mRNA were fractionated and analyzed by qPCR. Data are representative of one of two independent experiments (D–L). Each dot (I and K) represents one biological replicate. Dissociation-one phase decay is applied to fit KD (E). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (G and I–L), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F4.

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