Figure 6.
Overexpression of 4E-T blocks deadenylation-dependent decapping. (A) Schematic representation of the BGG-ARE reporter used in this study. A copy of the ARE sequence present in the 3′ UTR of the FOS mRNA was cloned downstream from the β-GLOBIN ORF. The FOS ARE is recognized by tristetraprolin (TTP) to promote target mRNA decay upon recruitment of the CCR4–NOT complex (Fabian et al. 2013; Bulbrook et al. 2018). (B,C) HEK293T cells were transfected with the BGG-ARE reporter and plasmids expressing λN-HA-MBP or λN-HA-TTP, V5-SBP-MBP, or V5-SBP-4E-T. The BGG-GAP reporter served as a transfection and normalization control. A representative Northern blot is shown in B. (A0) Deadenylated reporter mRNAs; (An) polyadenylated reporter mRNAs. (C) BGG-ARE mRNA levels were normalized to those of the BGG-GAP and set to1 in cells expressing λN-HA-MBP. Mean values ± SD are shown (n = 3). (*) P < 0.05, paired t-test. (D) Expression levels of the proteins used in B and C analyzed by Western blotting. TUBULIN served as a loading control. (E,F) Tethering of MS2-HA-NOT1 or MS2-HA-GFP to the BGG-6xMS2bs reporter in cells overexpressing V5-SBP-4E-T or V5-SBP-MBP. BGG-GAP was used as a transfection control. A representative Northern blot analysis is shown in E. A0, deadenylated and An, polyadenylated reporter mRNAs. (F) BGG-6xMS2bs mRNA levels were normalized to those of BGG-GAP and set to 1 in the experimental condition using MS2-HA-GFP and V5-SBP-MBP. Mean values ± SD are shown (n = 3). (*) P < 0.05; (ns) not significant, paired t-test. (G) The expression levels of the proteins used in E and F were verified using Western blotting. TUBULIN served as a loading control. Proteins were detected using anti-HA, anti-4E-T, anti-V5, and anti-TUBULIN antibodies.