Fig 2: IRAK2 mediates nuclear export of mRNAs of pro-inflammatory genes.(A) Heatmap showing the cytosolic and nuclear abundance of selected transcripts that require IRAK2 for nuclear export in response to LPS stimulation. Affymetrix microarray was performed to analyze the total mRNAs isolated from the cytoplasmic and nuclear fractions of 3 pairs of WT and IRAK2 knockout (KO) BMDMs treated with LPS for the indicated times. Transcripts-induced by LPS in the nuclei of both WT and KO cells were further ranked based on an index (I) that measures the impact of IRAK2 deficiency on transcript nuclear retention (See Materials and methods section). Scaled expression levels of transcripts encoding pro-inflammatory cytokines and chemokines are shown from blue to red, indicating low to high expression. (B) Total mRNAs isolated from the cytoplasmic and nuclear fractions of WT, IRAK2 KO and IRAK2 kinase-inactive (KI) BMDMs treated with LPS for the indicated times were subjected to RT-PCR analyses. (C) RNA-READ motif scanner, a regression-based framework which searches for previously defined RNA cis-motif was performed and the binding sites of SRSF1 (KGRWGSM, K: G/U; R:G/A; W:A/U; S:G/C; M:A/C) are significantly enriched in the 3’UTRs of the positive set versus the negative set (Materials and methods). (D) Confocal imaging of PLA (proximity ligation assay) signal of Hela cells transfected with HA-tagged SRSF1 together with FLAG-tagged IRAK2 WT, KK235AA, NLS mutant (K361/362/364A) and Sumo mutant (K123/182/592R). Mouse anti-HA and Rabbit anti-FLAG antibody were used for the proximity ligation assay. Green dots present PLA positive signal indicating the interaction of IRAK2 with SRSF1. Bar graph shows the percentage of PLA positive cells analyzed by Student’s t test. *p<0.05. Western blot analysis of Hela cells transfected with HA-tagged SRSF1 together with FLAG-tagged IRAK2 WT, KK235AA, NLS mutant (K361/362/364A) and Sumo mutant (K123/182/592R) with the indicated antibody. (E) Phosphorylation of SRSF1 by IRAK2 was assessed by in vitro kinase assay using recombinant IRAK2 and SRSF1. (F) WT and IRAK2 KI BMDMs treated with LPS for the indicated times, followed by immunoprecipitation (IP) with anti-SRSF1 antibody under denaturing condition (0.1% SDS in the lysis buffer) and analyzed by western blot analysis with the indicated antibodies. (G) WT, IRAK2 KO and IRAK2 KI BMDMs were treated with LPS for indicated times, followed by RNA immunoprecipitation with anti-SRSF1 antibody and RT-PCR analyses of the indicated mRNAs. The presented are the relative values normalized against IgG control (Materials and methods). The experiments were repeated for five times with similar results. Data represent mean ± SEM; *p<0.05 by Student’s t test.10.7554/eLife.29630.024Figure 3—source data 1.The numerical data for the graphs in Figure 3.(A) for Figure 3B. (B) for Figure 3D. (C) for Figure 3G.

Fig 3: IRAK2 kinase-inactive knock-in mice are resistant to LPS-induced septic shock.10 pairs Wild-type (WT) and IRAK2 kinase-inactive (KI) mice were injected intraperitoneally with 10 mg LPS/kg, and the survival of injected mice was monitored for 72 hr. (A) The survival curves were created by the method of Kaplan and Meier. (B) ELISA was performed for the levels of TNF and CXCL1 in the plasma 4 hr after LPS challenge. Data represent mean ± SEM; *p<0.05 by Student’s t test. (C) Model for the nuclear function of IRAK2. Upon LPS stimulation, IRAK2 is activated, followed by RanBP2-mediated sumoylation and subsequent nuclear translocation. Nuclear IRAK2 then phosphorylates SRSF1 and reduces SRSF1 binding to the target mRNAs which promotes the RNA binding of the nuclear export adaptor ALYREF and export receptor Nxf1 loading for export of mRNAs.10.7554/eLife.29630.036Figure 7—source data 1.The numerical data for the graphs in Figure 7.(A) for Figure 7A. (B) for Figure 7B.

Fig 4: SRSF1 binding renders the LPS-sensitivity of the target mRNAs for their nuclear export.(A) REMSA was performed to show binding of purified recombinant SRSF1 protein to the Cxcl1 3’UTR (nt 781–901), Cxcl1 3’UTR (nt Δ 790–840 in 781–901) and Cxcl1 3’UTR (800-855). (B–D) CMV-Luc- Cxcl1 3’UTR (721-940), (nt Δ 790–840 in 721–940) and (nt Δ 829–835 in 721–940) plasmids were transiently transfected in IRAK-deficient 293-IL1R (I1A) cells for 24 hr; followed by RNA immunoprecipitation with anti-SRSF1 antibody and RT-PCR analyses of the indicated mRNAs, the presented are the relative values normalized against IgG control (Material and methods) (B); Luciferase activity was measured (C); RT-PCR analysis of total mRNAs isolated from the cytoplasmic and nuclear fractions (D). (E) CMV-Luc- Cxcl1 3’UTR (721-940), (nt Δ 790–840 in 721–940) and (nt Δ 829–835 in 721–940) plasmid were transiently transfected in wild-type, IRAK2 KO and IRAK2 KI macrophages and treated with LPS for 12 hr, followed by luciferase assay. (F) For the transfected cells as described in Figure 6B–D, cell lysates were subjected to RNA immunoprecipitation with anti-ALYREF antibody and RT-PCR analyses of the indicated mRNAs. The presented are the relative values normalized against IgG control (Materials and methods). (G) REMSA was performed to show binding of purified recombinant ALYREF protein to the Cxcl1 3’UTR (nt 781–901), Cxcl1 3’UTR (nt Δ 790–840 in 781–901) and Cxcl1 3’UTR (800-855). Data represent mean ± SEM; *p<0.05 by Student’s t test.10.7554/eLife.29630.034Figure 6—source data 1.The numerical data for the graphs in Figure 6.(A) for Figure 6B. (B) for Figure 6C. (C) for Figure 6D. (D) for Figure 6E. (E) for Figure 6F.