Figure 2.

Assessment of conserved, MIR-derived target sites for let-7. (A) Let-7 target sites (yellow box) overlapping a MIR element (red boxes) annotated by RepeatMasker. MYO1F and E2F6 are two candidates where: (i) no let-7 site is present in the 3′ UTR aside from the MIR-derived site shown and (ii) PhyloP conservation scores (Mammal Cons track) showed sequence conservation coincident with the target site. (B) 3′ UTRs of MYCBP, MFSD4, E2F6 and MYO1F, each containing a single MIR-derived let-7 site, were cloned into dual-luciferase reporters and co-transfected into HEK293 cells with a synthetic let-7 mimic (Pre-miR™ at the doses indicated). Reactions were balanced to 1.0 nM with a non-targeting Pre-miR™ (ctrl). Luciferase activity is plotted as a percent of Renilla:Firefly ratio measured from the 0 nM let-7 (1.0 nM ctrl) treatment group. (C) HeLa cells, which express high levels of endogenous let-7a, were co-transfected with the luciferase reporters and a let-7a Anti-miR inhibitor (25, 50 nM), balanced to 50 nM with a negative control Anti-miR oligo (ctrl). Luciferase activity is presented as the Renilla:Firefly ratios measured 48 h post-transfection and normalized to the 50 nM ctrl dose of the corresponding reporter. N = 3 biological replicates with three technical replicates per assay; error bars = SD. *P ≤ 0.05 (Student's t-test; two-tailed). (D) Cumulative distribution functions are shown summarizing gene expression changes (log₂ fold change) in response to let-7 over-expression (si-let-7), relative to the control siRNA (si-GFP). Transcripts were grouped according to annotations of 3′ UTR-MIRs and let-7 target sites, as indicated in the figure legends. (E) As in D, except gene expression changes were measured in response to let-7 inhibition (let-7 2′OMe) relative to a control oligo (Luc. 2′-OMe).