Figure 4

The Lif 3′UTR contains conserved regions with AREs that influence RNA levels in rMC-1 cells. A) Sequence alignment of the 3,180 bp long Lif 3′UTR sequences from Mus musculus (Mm), Homo sapiens (Hs), Rhesus monkey (Rm), Canis lupus familiaris (Cf), Bos taurus (Bt) and Pan troglodytes (Pt) revealed two highly conserved AU-rich regions (R I and R II) and seven smaller regions containing AREs (R27, R31, R32, R35, R37), a miRNA binding site (R38) or both (R36). B, C) rMC-1 cells were transfected with luciferase-reporter constructs, as indicated. Luminescence levels were determined after 24 hours and expressed relative to a construct with a minimal 3′UTR (ΔUTR). D) rMC-1 cells were transfected with luciferase-reporter constructs carrying R36, R27, R32 or ΔUTR sequences. Gene expression was blocked with ActD 24 hours after transfection. Luminescence levels were determined at 0 to 4 hours after ActD addition and shown relative to the levels of the ΔUTR control construct that were set to 1 for each time point. Shown are means ± SEM of N = 4 to 5. One-way ANOVA (B, C) or two-way ANOVA (D) with Dunnett’s posttests were used to compare constructs to the levels of the ΔUTR construct. (*) P <0.05, (**) P <0.01, (***) P <0.001 and (****) P <0.0001. ANOVA, analysis of variance; AREs, AU-rich elements; SEM, standard error of the mean.