Both translation reinitiation and uAUG leaky scanning are involved in the translational initiation at the main AUG codon. (A) Schematic representation of reporter constructs. The pGL2-WT plasmid contains the wild-type human EPO 5′-leader sequence; the pGL2-no_uSTOP construct presents the EPO 5′-leader sequence with a mutation (UGA→AGA) at the uORF translation termination codon, which makes the uORF overlap with the luciferase ORF (the cross represents the point mutation); and the pGL2-optimal_uAUG contains the EPO 5′-leader sequence with an optimal uAUG sequence context (gggAUGa→gccAUGg; represented by a bold lined box). (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.
