CIKS/DDX3X interaction controls the stability of the Zc3h12a mRNA induced by IL-17

Abstract

IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.

Figure 1. Physical interaction between CIKS and DDX3X

(A) Anti-FLAG immunoprecipitates (IP) of HEK293 cell extracts transfected with FLAG-CIKS and HA-DDX3X were Western-blotted with anti-HA antibodies to detect coimmunoprecipitated DDX3X (Top panel). (Middle and Bottom panels) Western blots with anti-HA (anti-DDX3X) and anti-FLAG (anti-CIKS) antibodies on whole-cell extracts are shown, respectively. (B) Mapping of the DDX3X-binding domain on CIKS. HEK293 cells were transfected with HA-DDX3X and FLAG-CIKS or different FLAG-CIKS deletion mutants. Cell extracts were immunoprecipitated with anti-FLAG antibodies (CIKS) followed by Western blot (WB) anti-HA (DDX3X). The presence of -HA and -FLAG proteins in the whole cell extract is shown in the middle and bottom panels, respectively. (C) Mapping the CIKS-binding domain on DDX3X. HEK293 cells were transfected with FLAG-CIKS and HA-DDX3X or different HA-DDX3X deletion mutants. Cell extracts were immunoprecipitated with anti-FLAG antibodies (CIKS) followed by Western blot (WB) anti-HA (DDX3X). The presence of -HA and -FLAG proteins in the whole cell extract is shown in the middle and bottom panels, respectively. (D) Interaction between CIKS and endogenous DDX3X. CIKS^−/− and reconstituted FLAG-CIKS MEFs, were treated for the indicated period of time with IL-17 (200ng/ml). Cell extracts were immunoprecipitated with anti-FLAG antibodies and Western blotted (WB) with anti-DDX3X antibodies.

Figure 2. DDX3X controls the half-life of the Zc3h12a mRNA

Control MEF (siCo), DDX3X-depleted MEF (siDDX3X) and CIKS^−/− MEF were treated with TNF-α (2000U/ml) for 60 minutes, and then with IL-17 (200ng/ml) and actinomycin D (5µg/ml). After additional 90 min RNA was extracted and the abundance of the Zc3h12a (C), Cxcl1 (D), Cxcl5 (E), Il-6 (F) was measured by Real-Time PCR. (G) Time course analysis of Cxcl1 and Zc3h12a abundance after treatment with TNF-α (60 minutes) followed by the combined addition of IL-17 and actinomycin D. Expression of DDX3X protein (A) and mRNA (B) in control (siCo) and DDX3X interfered (siDDX3X) MEFs is shown. Data are representative of three independent experiments. Statistical analysis by unpaired Student’s t-test: * p<0.05, ** p<0.01. N.S. not statistically significant.

Figure 3. IKKε interacts with DDX3X and controls the Zc3h12a mRNA stability

(A) Anti-Myc immunoprecipitates (IP) of HEK293 cell extracts transfected with Myc-IKKε, FLAG-CIKS and HA-DDX3X were Western-blotted with anti-HA antibodies to detect coimmunoprecipitated DDX3X (Top panel). Western blots with anti-HA (DDX3X) and anti-FLAG (CIKS) and anti-Myc (IKKε) antibodies on whole-cell extracts are shown. * Indicates non-specific cross-reactive bands. B) Interaction between CIKS and endogenous IKKε and DDX3X. CIKS^−/− and reconstituted FLAG-CIKS MEFs, were treated for the indicated period of time with IL-17 (200ng/ml). Cell extracts were immunoprecipitated with anti-FLAG antibodies and Western blotted (WB) with anti-IKKε and with anti-DDX3X antibodies. Expression of IKKε protein (C) and mRNA (D) in control (siCo) and IKKε-interfered (siIKKε) MEFs. (E) Zc3h12a mRNA level in control (siCo) and IKKε-interfered MEFs (siIKKε), after treatment with TNF-α (60 minutes) followed by combined addition of IL-17 and actinomycin D. Data are representative of three independent experiments. Statistical analysis by unpaired Student’s t-test: * p<0.05, ** p<0.01.

Figure 4. TRAF2 and TRAF5 are required for IL-17-induced stabilization of the Zc3h12a mRNA

Expression of TRAF2 (A, B) and TRAF5 (D, E) in control (siCo), TRAF2 (siTRAF2) and TRAF5 (siTRAF5) interfered MEFs. Zc3h12a mRNA level in control (siCo), TRAF2 (siTRAF2) (C), TRAF5 (siTRAF5) (F) and double interfered (siTRAF2/5) (G) MEFs, after treatment with TNF-α (60 minutes) followed by combined addition of IL-17 and actinomycin D for the indicated period of time. Data are representative of three independent experiments. Statistical analysis by unpaired Student’s t-test: * p<0.05, ** p<0.01.

Figure 5. TRAF6 is not required for IL-17-induced stabilization of the Zc3h12a mRNA

Expression of TRAF6 (A, B) in control (siCo), and TRAF6-interfered (siTRAF6) MEFs. (C) Zc3h12a mRNA level in control (siCo), and siTRAF6 MEFs, after treatment with TNF-α (60 minutes) followed by combined addition of IL-17 and actinomycin D. (D) Expression of CIKS E17A in CIKS^−/− MEFs reconstituted with CIKSE17A. (E) Zc3h12a mRNA level in CIKS^−/− MEFs, reconstituted with CIKS or CIKS E17A after treatment with TNF-α (60 minutes) followed by combined addition of IL-17 plus actinomycin D for the indicated period of time. Data are representative of three independent experiments. Statistical analysis by unpaired Student’s t-test: ** p<0.01.

Figure 6. IL-17 modulates the binding of DDX3X to the Zc3h12a mRNA

A) HeLa cells were transfected with an expression vector encoding FLAG-DDX3X or with an empty vector. Twenty-four hours after transfection, cells were treated with TNF-α (2000U/ml) plus IL-17 (200 ng/ml) for two hours, and DDX3X was immunoprecipitated by using anti-FLAG antibodies. RNA was extracted from the immunoprecipitates, reverse transcribed, and Zc3h12a, Il6 and cxcl1 were amplified by PCR. The presence of transfected DDX3X in the whole cell lysate and in the immunoprecipitates is shown. B) Il6 mRNA level in control (siCo) and DDX3X-interfered MEFs (siDDX) after treatment with TNF-α (2 hours) or TNF-α (2 hours) followed by IL-17 for additional 8 hours. Statistical analysis by unpaired Student’s t-test ** p<0.01.