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. 2018 Feb 7;7:e35034. doi: 10.7554/eLife.35034

Figure 3. 4EHP enables miRNA-mediated silencing of Dusp6 mRNA.

(A) RIP analysis of the association of eIF4E with Dusp6 mRNA in WT and 4EHP-KO MEFs. eIF4E was immunoprecipitated using a monoclonal antibody. Levels of the indicated mRNAs (normalized to β-actin mRNA) in the inputs and eIF4E-bound mRNAs were analyzed by RT–qPCR. Data are mean ± SD (n = 3). (B) Top; Schematic representation of the psiCHECK-FL-Dusp6 3´ UTR reporter. Bottom; CTR, CNOT1, 4E-T, or 4EHP-knockdown cells were co-transfected with psiCHECK-FL-Dusp6 3´ UTR reporter or the psiCHECK reporter (as control) in HEK293T cells. Luciferase activity was measured 24 hr after transfection. Firefly (F-Luc) values were normalized against Renilla (R-Luc) levels, and repression fold was calculated for the psiCHECK-FL-Dusp6 3´ UTR reporter relative to psiCHECK reporter level for each condition. Data are mean ± SD (n = 3). (C) The psiCHECK reporter (control) or psiCHECK-RL with truncated fragments of the Dusp6 3´ UTR were transfected into the HEK293T cells. Luciferase activity was measured 24 hr after transfection. R-Luc values were normalized against F-Luc levels, and repression fold was calculated for the psiCHECK-RL-Dusp6 3´ UTR reporter relative to psiCHECK reporter level for each condition. Data are mean ± SD (n = 3). (D) WB for the indicated proteins in U251 cells transfected with si4EHP or the indicated miRNA inhibitors. (E) The psiCHECK reporter (control) or psiCHECK-FL-Dusp6 3´ UTR were co-transfected along with the mock or miR-145 inhibitor in the control (CTR) or 4EHP-KO HEK293 cells. Luciferase activity was measured 24 hr after transfection. F-Luc values were normalized against R-Luc levels, and repression fold was calculated relative to the psiCHECK reporter/control inhibitor for each condition. Data are mean ± SD (n = 3). The p-values was determined by two-tailed Student's t‐test: (ns) non-significant, (*) p<0.05; (**) p<0.01; (***) p<0.001.

Figure 3.

Figure 3—figure supplement 1. Repression of DUSP6 expression by CCR4-NOT complex.

Figure 3—figure supplement 1.

(A) WB for the indicated proteins in control or siRNA transfected U251 cells. (B) Diagram of Dusp6 mRNA 3´ UTR, predicted miRNA binding sites, pumilio responsive element (PRE), and truncation fragments of the UTR created for cloning into the reporter construct used in Figure 3C. (C) RNA stability assay of Dusp6 mRNA in Mock and miR-145-inhibitor transfected cells. The quantity of RNA at different time points was determined by RT-qPCR. Values are normalized to 28S rRNA. Data are mean ± SD (n = 3). (D) Sequence alignment of 10 cloned PCR products amplified from the genomic segment of 4EHP targeted by 5´-TGAGCTCGTGGGACGGCCGG sgRNA showing the disruption of the coding sequence (related to Figure 3E).