Figure 4. The S513A mutation destabilizes Regnase-1 protein but does not affect target mRNA abundance.

(A–C) Immunoblot analysis of Zc3h12a^WT/WT and Zc3h12a^S513A/S513A MEFs stimulated with IL-1β (10 ng/ml) (A), BMDMs stimulated with LPS (100 ng/ml) (B), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) (C) for indicated time. PECs were pretreated with MG-132 (5 μM) 2 hr before the stimulation. (D)-(F) mRNA expression of Zc3h12a and Il6 in Zc3h12a^WT/WT and Zc3h12a^S513A/S513A MEFs stimulated with IL-1β (10 ng/ml) for 4 hr (D), BMDMs stimulated with LPS (100 ng/ml) for 4 hr (E), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time (F). (G)-(I) IL-6 secretion in Zc3h12a^WT/WT and Zc3h12a^S513A/S513A MEFs stimulated with IL-1β (10 ng/ml), IL-17A (50 ng/ml), or TNF (10 ng/ml) for 24 hr (G), BMDMs stimulated with Pam₃CSK₄ (1 or 10 ng/ml), poly I:C (10 or 100 μg/ml), LPS (10 or 100 ng/ml), R848 (10 or 100 nM), or CpG DNA (0.1 or 1 μM) for 24 hr (H), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml), R848 (100 nM), or IL-1β (10 ng/ml) for 24 hr (I). (J) Schematic representation of Model 1 in which 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs. This model could explain the experimental observations. (K) Schematic representation of Model 2 in which 14-3-3-bound Regnase-1 maintains some ability to degrade its target mRNAs. This model is not consistent with the experimental observations. In (D)-(I), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. Data is representative of two independent experiments, each with three biological replicates.

Figure 4—figure supplement 1. Schematic illustration of Zc3h12a gene in mice.

The result of Sanger sequencing around S513 of Regnase-1 are shown below.

Figure 4—figure supplement 2. The protein stability of Regnase-1-WT and S513A.

Immunoblot analysis of Zc3h12a^WT/WT and Zc3h12a^S513A/S513A thioglycollate-elicited PECs treated with LPS (100 ng/ml) and CHX (100 μg/ml) for indicated time. CHX and LPS were added at the same time.

Figure 4—figure supplement 3. Il6 expression in Zc3h12a^–/– PECs mRNA expression of Il6 and Zc3h12a in Zc3h12a^WT/WT and Zc3h12a^–/– thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time.

Bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation.

Figure 4—figure supplement 4. The stability of Regnase-1 target mRNAs.

The remaining mRNA levels of indicated genes in Zc3h12a^WT/WT and Zc3h12a^S513A/S513A MEFs. Actinomycin D (ActD, 10 μg/ml) was added after 4 hr stimulation with IL-1β (10 ng/ml). Dots represent mean values of biological replicates (n = 3), and error bars represent standard deviation.

Figure 4—figure supplement 5. S513A mutation of Regnase-1 does not affect gene expression.

Transcriptome analysis of Zc3h12a^WT/WT and Zc3h12a^S513A/S513A thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time. Several known Regnase-1 target transcripts are annotated. None of transcripts shows significant (adjusted p value < 0.05) difference between Zc3h12a^WT/WT and Zc3h12a^S513A/S513A.