Human miR-27a* specifically targets both Prf1 and GzmB 3′ UTR sequence. (A top) Predicted miR-27a* binding sites. (Bottom) Schematic diagrams of Prf1 and GzmB 3′ UTRs (wild type [WT]) or their deletions (D1 and D2) and mutants (M). Point mutations are bolded. Numbers indicate positions of nucleotides in the 3′ UTRs. Crosses indicate the mutation depicted in the top panel. (B-D) Reporter assay using IB analysis. HEK-293FT cell lines were cotransfected with combinations of reporter plasmids containing WT, D, or M forms of Prf1 and GzmB 3′ UTRs and miR-27a* or its mutated miRNA (miR-27a*M). Reporter AANAT mRNA levels were normalized to Neo^R mRNA level as an internal control of the vector. Net amount of translated AANAT protein was determined by IB. GAPDH served as loading control. Data are representative of 4 independent experiments (mean ± SEM of triplicates).