Deletion of bantam sites identified by target prediction programs incompletely derepresses natural hid reporter construct (R-Luc-hid). (A) Schematic of predicted bantam binding sites in hid 3′ UTR from commonly used algorithms and with an extended seed definition. Deletion of four bantam seed elements according to TargetScan (red squares) yields R-Luc-hidΔ4 whereas a deletion of eight potential seed sites (including blue squares based on an extended seed definition) yields R-Luc-hidΔ8. (B) R-Luc-hid seed deletion constructs exhibit differential expression levels of luciferase activity. R-Luc-hid, R-Luc-hidΔ4, or R-Luc-hidΔ8 reporter plasmids were transfected (5 ng/well) and for each reporter construct, the normalized Renilla/firefly luciferase ratio was divided by the normalized Renilla/firefly value of R-Luc-hid.
