FIG 5.

miR-K3 targets 3′UTRs of ORF31, ORF32, and ORF33 through binding sites in the ORF33 coding region. (A) Schematic illustration of the ORF30-33 gene locus, their transcripts, 3′UTR luciferase reporters, and constructs expressing 3×FLAG fusion proteins. S1 and S2 are predicted as miR-K3-5p binding sites located in the coding region of ORF33. For the 3×FLAG fusion protein constructs, each ORF, including the downstream 3′UTR sequence, is fused in frame with the N terminus of the 3×FLAG tag. (B) Alignment of miR-K3-5p with predicted binding sites in ORF30-33 transcripts. Mutated nucleotides in the binding sites are shown in red type. Solid lines indicate the Watson-Crick base pairing, and dotted lines indicate wobble base pairing. (C) miR-K3 represses ORF31, ORF32, and ORF33 but not ORF30 3′UTRs by targeting two binding sites (S1 and S2) residing in the ORF33 coding region. miRNA expression plasmid or a miRNA expression control vector was cotransfected with the wild-type 3′UTR reporter, mutant 3′UTR reporter, or a reporter control vector into 293T cells, together with a pSV-β-galactosidase plasmid for 48 h, and the relative luciferase activities were examined and normalized for β-galactosidase activity. Experiments were repeated three times, and the results are presented as averages and STD by setting the values of miRNA expression vector control as “1”. (D and E) The mimic of miR-K3-5p does not repress the expression of fusion protein from the 3×FLAG-ORF30 construct (D). The mimic, but not the scrambled control mimic, of miR-K3-5p inhibits the expression of fusion proteins of ORF31, ORF32, and ORF33 by targeting S1 and S2 binding sites (E). Western blot analyses of 3×FLAG-ORF30 fusion protein (D) and of 3×FLAG-ORF31, -ORF32, and -ORF33 fusion proteins (E) in 293T cells were performed. The mimic of miR-K3-5p or a scrambled control mimic was cotransfected with each FLAG-tagged protein expression construct or its mutants with mutation in S1, S2, or both binding sites into 293T cells for 48 h, and the expression of the fusion protein was detected by Western blotting. A 3×FLAG-tagged firefly luciferase construct was also cotransfected as an internal control.