The Repertoire and Features of Human Platelet microRNAs
Figure 6
Redirection of platelet miR-140-3p targeting upon a 1-nt shift of the 5′ cleavage site.
(A) mRNA target prediction for the reference mature miR-140-3p sequence and the miR-140-3p isomiR harboring a 1-nt 5′ shift using TargetScan. The reference isomiR potentially regulates 240 different mRNA targets, whereas the shifted isomiR potentially regulates 367 mRNA targets. Notably, only 50 of these mRNAs are predicted to be regulated by both forms of miR-140-3p. (B) Experimental validation of the target specificity of the 1-nt 5′ shifted isomiR for CAP1 mRNA. HEK 293 cells were co-transfected with miR-140 microRNA duplexes, containing either the reference microRNA sequence (Reference) or the most abundant 1-nt 5′ shifted isomiR (Shifted), and a reporter gene construct in which the adenylate cyclase-associated protein 1 (CAP1) 3′UTR was inserted downstream of the Rluc reporter gene. Base pairing complementarity between miR-140-3p microRNAs and their binding sites is shown in the upper panel. Base pairing involving the microRNA seed region is highlighted in color. The blue X denotes the loss of base pairing of nt 2 of the miR-140-3p reference isoform, which may explain its lower efficiency in regulating CAP1 mRNA 3′UTR expression. Rluc and Fluc activities were measured, and the values were normalized to those obtained with a non-relevant RNA duplex (n = 3 to 4 experiments, in duplicate) (lower panel). ** p<0.01, *** p<0.001 (Tukey-Kramer Multiple Comparisons Test).