bantam miRNA-mediated translational repression of synthetic reporter mRNAs in D. melanogaster S2 cells. (A) Schematic representation of reporter constructs carrying synthetic 3′ UTRs. (B) Differential expression activity of R-Luc-synthetic and R-Luc-flipped constructs is dose dependent. A range, 100 ng/well to 0.5 ng/well, of R-Luc reporter DNA plasmids was tested in 24-well plates while keeping the internal transfection control F-Luc plasmid constant. Normalized Renilla/firefly activity was measured for each construct with fold repression representing the ratio of R-Luc-flipped/R-Luc-synthetic. (C) Differential expression of R-Luc-synthetic and R-Luc-flipped reporters is specifically mediated by bantam miRNA. R-Luc-synthetic reporter DNA plasmid was co-transfected with 100 nM 2′-O-methylated RNA oligonucleotides (anti-miRNA) complementary to one of several endogenous D. melanogaster miRNAs (bantam, miR-1, miR-184, miR-6) or to the C. elegans lin-4 miRNA. Renilla luciferase values (normalized to firefly values) were normalized relative to the control +anti-lin-4 sample. (D) Titration of R-Luc-synthetic construct with anti-bantam RNA oligonucleotides yields maximal derepression that mirrors reporter titration in A. For each panel, values shown represent the mean ± SD from three independent experiments.
