Translation attenuates, but does not block, miR-K5-mediated down-regulation. (A) Experimental constructs of the miR-K5 site embedded within the extended Renilla LUC (Rluc) in the isogenic dual luciferase reporter. VEC contains no ORF56 3′UTR. 3′UTR and C-ORF have an identical sequence, except at three nucleotides that disrupt the UAA stop codon of Rluc and two additional stop codons (not shown in the figure) preceding the miR-K5 site, thereby allowing extension of the ORF to 20 nucleotides 3′ to the miR-K5 site. 3′UTR-tK5 and C-ORF-tK5 are isogenic derivatives in which the miR-K5 site is mutationally inactived. “Stop” denotes stop codon. The miR-K5 site is in blue and mutated nucleotides are in red. (B) HEK293 cells were cotransfected with the indicated reporter constructs and miRNAs. Mean values and error bars (SD) were derived from quadruplicate independent transfections. Statistical significance is indicated (p value, two-tailed t test).