eIF6 prevents the nuclear export of cytokine mRNAs by inhibiting the methyltransferase activity of Nsun2 in ILC3s. (A and B) Western blot analysis of the co-IP between p-p38α and Nsun2 (A) or between eIF6 and Nsun2 (B) in MNK-3 cells treated with or without IL-1β. (C) Representative images of immunofluorescent staining of eIF6 (green), Nsun2 (red), and smFISH of Il22 mRNA (cyan) in MNK-3 cells stimulated with or without IL-1β and SB203580. Scale bars, 10 μm. (D) The Nsun2-mediated m5C assay with purified eIF6 and Nsun2 protein was conducted in vitro. The Il22 (red dot) or Csf2 (blue dot) mRNA was synthesized in vitro. Nsun2-mediated m5C modification on the cytokine transcripts with Nsun2 (red) or with Nsun2 plus eIF6 (blue) was performed. Then the mRNA was purified and digested to single nucleotide. The m5C modification was determined by HPLC-MS (left). The ratios of methylated cytosine to unmethylated cytosine were shown (right). Data are pooled from two independent experiments. (E) RNA dot blot analysis of mRNAs with m5C modification in control or eIF6-knocked-down MNK-3 cells with or without IL-1β stimulation. (F) The potential eIF6 (green)-Nsun2 (purple) interaction pattern predicted by AlphaFold2. The SAM binding pocket (gray) of Nsun2 and the interactive surface (ball and stick presented) are shown. The dash lines between amino acids indicate the hydrogenic bone of Nsun2/eIF6 complex. (G) The binding affinity between wild type eIF6 (eIF6WT) and Nsun2 was measured by Biacore SPR assay. eIF6WT was attached to the CM-5 chips and the indicated Nsun2 flow phase was delivered to the Biacore system. (H) The binding affinity between Nsun2 and SAM was measured by Biacore SPR assay. Nsun2 was attached to the CM-5 chips and the indicated SAM flow phase was delivered to the Biacore system. (I) The binding affinity between Nsun2 (fixed phase) and SAM in the presence of eIF6 was measured by Biacore SPR assay. (J) The binding affinity between eIF6WT (fixed phase) and Nsun2 in the presence of SAM was measured by Biacore SPR assay. (K) The binding affinity between mutant eIF6EN-AG (fixed phase) and Nsun2 was measured by Biacore SPR assay. (L) The control and WT or mutant eIF6-overexpression MNK-3 cells were activated and the cytokine production was analyzed by flow cytometry. Data are representative of one of two independent experiments (A–E and G–L). Each dot (D and L) represents one biological replicate. Biacore Insight Evaluation software (GE Healthcare) is applied to calculate the KD (G–K). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (L), two-tailed Student’s paired t test (D), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F5.
Figure 5.

eIF6 prevents the nuclear export of cytokine mRNAs by inhibiting the methyltransferase activity of Nsun2 in ILC3s. (A and B) Western blot analysis of the co-IP between p-p38α and Nsun2 (A) or between eIF6 and Nsun2 (B) in MNK-3 cells treated with or without IL-1β. (C) Representative images of immunofluorescent staining of eIF6 (green), Nsun2 (red), and smFISH of Il22 mRNA (cyan) in MNK-3 cells stimulated with or without IL-1β and SB203580. Scale bars, 10 μm. (D) The Nsun2-mediated m5C assay with purified eIF6 and Nsun2 protein was conducted in vitro. The Il22 (red dot) or Csf2 (blue dot) mRNA was synthesized in vitro. Nsun2-mediated m5C modification on the cytokine transcripts with Nsun2 (red) or with Nsun2 plus eIF6 (blue) was performed. Then the mRNA was purified and digested to single nucleotide. The m5C modification was determined by HPLC-MS (left). The ratios of methylated cytosine to unmethylated cytosine were shown (right). Data are pooled from two independent experiments. (E) RNA dot blot analysis of mRNAs with m5C modification in control or eIF6-knocked-down MNK-3 cells with or without IL-1β stimulation. (F) The potential eIF6 (green)-Nsun2 (purple) interaction pattern predicted by AlphaFold2. The SAM binding pocket (gray) of Nsun2 and the interactive surface (ball and stick presented) are shown. The dash lines between amino acids indicate the hydrogenic bone of Nsun2/eIF6 complex. (G) The binding affinity between wild type eIF6 (eIF6WT) and Nsun2 was measured by Biacore SPR assay. eIF6WT was attached to the CM-5 chips and the indicated Nsun2 flow phase was delivered to the Biacore system. (H) The binding affinity between Nsun2 and SAM was measured by Biacore SPR assay. Nsun2 was attached to the CM-5 chips and the indicated SAM flow phase was delivered to the Biacore system. (I) The binding affinity between Nsun2 (fixed phase) and SAM in the presence of eIF6 was measured by Biacore SPR assay. (J) The binding affinity between eIF6WT (fixed phase) and Nsun2 in the presence of SAM was measured by Biacore SPR assay. (K) The binding affinity between mutant eIF6EN-AG (fixed phase) and Nsun2 was measured by Biacore SPR assay. (L) The control and WT or mutant eIF6-overexpression MNK-3 cells were activated and the cytokine production was analyzed by flow cytometry. Data are representative of one of two independent experiments (A–E and G–L). Each dot (D and L) represents one biological replicate. Biacore Insight Evaluation software (GE Healthcare) is applied to calculate the KD (G–K). Data are mean ± SEM and statistical significance was tested by two-tailed Student’s unpaired t test (L), two-tailed Student’s paired t test (D), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F5.

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