Fig. 2.

Ago2 RNase activity is required for RNAi enhancement at perfectly matched targets. (A) 293 cells were transfected with a firefly luciferase construct containing different let-7a binding sites in its 3′ UTR (pm, perfect match; mm, two mismatches in middle of miRNA-binding site; seed mm, two mismatches in seed region of miRNA–3′UTR interaction site; 1x, one binding site; 4x, four tandem binding sites), renilla luciferase and expression plasmids for let-7a or/and Ago2. Depicted is the mean fold inhibition (+SEM) of firefly luciferase activity compared with a transfection neither containing the ectopic miRNA nor Ago2 (ctrl). Let-7a only weakly inhibited luciferase activity, whereas Ago2 enhanced it. RNAi specificity was conserved because neither mismatch nor seed mutant controls were affected at all by Ago2 expression. (B) RNase deficient Ago2 mutants (D597A, D669A) failed to enhance inhibition of luciferase activity by let-7a in 293 cells. (C) Ago2 cotransfection into 293 cells also enhanced the inhibition of let-7a siRNA transfected as double-stranded siRNA oligonucleotide. (D) Efficiency of miR-143 toward a luciferase construct containing perfectly matched miR-143 binding sites in its 3′UTR was also enhanced by Ago2 cotransfection into 293 cells.