PUM1 and PUM2 positively regulate FOXP1 mRNA expression via two 3′UTR located PBE. (A) Schematic representation of human FOXP1 mRNA with the 2 PUM-binding elements (PBE), PBE1 and PBE2, boxed (red), and regions A, B, C delineated. The numbers indicate positions in 3′UTR, starting from translation stop codon. (B) RNA pull-down assay. MOLM-14 cell lysates were incubated with WT₁ or MUT₁ biotin-labeled FOXP1-PBE1 probes, and none or ×5 unlabeled PBE1 (WT₁ or MUT₁) or PBE2 (WT₂ or MUT₂) competitor riboprobes, as indicated. Pulled‐down PUM proteins were analyzed by immunoblotting. (C) RNA immunoprecipitation assays. Lysates from shRNA-transduced MOLM-14 cells were immunoprecipitated (IP) using control (IgG), PUM1 or PUM2 antibody, as indicated. Upper panel: Immunoblot analysis of the indicated proteins. Lower panel: RT-qPCR analysis of FOXP1 mRNA present in the IPs. The results are expressed as percent of input (n = 3). (D) Luciferase assay. MOLM-14 cells were transfected with control (siCtrl), PUM1 (siPUM1), or PUM2 (siPUM2) siRNA, and with the indicated luciferase reporter construct harboring the FL FOXP1-3′UTR. Relative luciferase activity was monitored 24 hours later and expressed as the ratio between Renilla luciferase (Rluc) activity and Firefly luciferase (Fluc) activity, used as transfection control. Data are expressed relative to expression in FOXP1-3′UTR FL and siCtrl transfected cells (n = 4). (E) Luciferase assays were performed as in panel D, with luciferase constructs harboring the indicated regions of the FOXP1-3′UTR (A-C) with WT, or doubly MUT1/2 PBEs. Data are expressed relative to expression in WT-FOXP1-3′UTR-C and siCtrl transfected cells (n = 4). (F) Luciferase assay. MOLM-14 cells were transfected with FOXP1-3′UTR-C luciferase reporter constructs harboring WT PBE1 and PBE2, mutated PBE1 (MUT1), mutated PBE2 (MUT2), or doubly mutated PBE1 and PBE2 (MUT1/2). Relative luciferase activity was monitored 24 hours later, expressed as the ratio Rluc/Fluc, and compared relative to expression in WT-FOXP1-3′UTR-C transfected cells (n = 4). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).