Fig. 8

Regnase-1 directly destabilizes AEC-specific target mRNAs. a RNA-immunoprecipitation (RIP)-qPCR analysis of indicated genes in the lung of WT or Reg1-FLAG-Knock-in (Reg1-FLAG-KI) mouse. Relative enrichment of mRNA amount compared with input sample is shown. Each dot indicates one sample. b Tet-off assay to analyze the altered stability of Ccl28 or Pigr mRNA in the presence of Regnase-1. HEK293 tet-off cells were co-transfected with either of WT or D141N mutant Regnase-1-expressing plasmid or control (empty) plasmid together with pTREtight-expressing CDS plus 3′UTR sequences of the genes indicated. The amount of remaining RNA was evaluated by RT-qPCR, and the amount at 2 h was compared. Each value indicates the average of three independent experiments. c Luciferase reporter assays for the evaluation of 3′UTR-dependent suppression of gene expression by Regnase-1. HEK293 cells were co-transfected with WT or D141N mutant Regnase-1-expressing plasmid or control (empty) plasmid together with pGL3-promotor plasmid with the 3′UTR sequence of the indicated genes, and luciferase activity was evaluated 48 h after the transfection. Data are representative of two independent experiments with n = 3. Data are shown as mean ± s.d. Two-tailed Student’s t-test was used for statistical analyses (b, c)