3′ LIFE assay identifies 3′ UTRs repressed by let-7c and miR-10b. (A) Summary of 3′ LIFE screens. We tested 1407 3′ UTRs in triplicate for targeting by each miRNA in cotransfection experiments in HEK293T cells (∼13,000 dual luciferase reactions). A 3′ UTR was considered a putative target if it exhibited >20% repression and P < 0.05. The top 30 repressed genes for each miRNA are listed adjacent to each heatmap, with the type(s) of miRNA target site(s). Predicted targets, both deeply and poorly conserved, were identified using TargetScan 7.0. (B) Characteristics of miRNA target sites in 3′ UTR library separated by target score measured by the 3′ LIFE assay. (C) Alignments of target sites from the top 50 hits with a canonical seed for let-7c (left) and miR-10b (right). Asterisks represent statistically significant nucleotides (P < 0.05, χ²). (D,E) Distance of putative miRNA targets to the mRNA end. 3′ UTRs in the library with one canonical miRNA target site and one canonical polyadenylation sequence (AAUAAA) were analyzed for the relative distance between the poly(A) site and the miRNA target site. Genes were separated based on targeting status.