The 3′ UTR of the EPO mRNA has no influence in the relative inhibitory effect of the uORF. (A) Schematic of the FLuc reporter constructs containing the native luciferase 3′ UTR (pGL2-Luc) or the 3′ UTR sequence (dark gray box) of the human EPO transcript (pGL2-Luc-3′ UTR). (B) HEK293, HepG2, and HeLa cells were transiently cotransfected with each one of the constructs described in A and with a plasmid encoding RLuc (pRL-TK) and analyzed as described in the legend to Figure 2B. (C) Schematic of the FLuc reporter constructs containing the human EPO 5′-leader sequence with the intact uORF and the luciferase 3′ UTR (pGL2-WT), pGL2-WT-3′ UTR (dark gray box), or the EPO 5′-leader sequence with a disrupted uORF due to the uAUG→UUG mutation (represented by a cross) and the EPO 3′ UTR sequence (dark gray box; pGL2-no_uAUG-3′ UTR). (D) HEK293, HepG2, and HeLa cells were transiently cotransfected with each one of the constructs described in C and with a plasmid encoding RLuc (pRL-TK) and analyzed as described in the legend to Figure 2B.
