Fig. 4.

Direct TP53INP1 targeting by and possible co-evolution with miR-524-5p. a Phylogenetic tree alignment (top panel) and sequence comparison (bottom panel) of the 3’-UTR of TP53INP1 ortholog transcript sequences in different species. b Identification of four putative miR-534-5p target sites (red vertical bars) in the 3’-UTR of the human TP53INP1 transcript. c Experimental validation of miR-524-5p targeting TP53INP1 in luciferase assays. In the top panel, alignment of miR-524-5p with the putative target site 2 (boxed and in bold letters) in the 3’-UTR of the TP53INP1 transcript (see text) is shown, so are the mutations (in italics and underlined) in the luciferase construct Mut2. HCT-15 transfected with the wild-type (WT2) or the mutant (Mut2) luciferase constructs alone, or in the presence of the miR-524-5p mimic or a validated negative control (NC), was performed before luciferase assays. The data shown were derived from two independent experiments in triplicate. *p < 0.05. d Alignment of sequences around the active target site 2 of TP53INP1 in different species. The miR-524-5p miRNA sequence is shown in blue above the TP53IPN1 sequences. The targeted core sequences are underlined and shown in bold. Similar nucleotides relative to the human gene are shown in italics and in bold letters. CDS coding sequence.