Figure 3.

The U3-derived fragments behave like low proficiency miRNAs in reporter assays. (A) Schematic overview of the reporter construct used for analysing miRNA function in human cells. (B) The relative expression of CFP and YFP in individual cells (dots) was determined by flow cytometry in cells containing constructs encoding no miRNA target site (Empty), a sequence not targeted by any known human miRNA (Non-cognate) or the indicated miRNA target sites in one or three copies, within the 3′ UTR of the CFP gene. Grey lines indicate fitting to a previously described model of miRNA-mediated regulation (27,29). The significance of differences between cells transfected with the empty construct and those carrying the miRNA target sites indicated was calculated using the Mann-Whitney-Wilcox test (ns – not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). (C) The relative expression of CFP and YFP in individual cells (dots) was determined by flow cytometry, as a measure of AGO2 slicing activity, in cells containing empty reporter constructs (Empty) or reporter constructs encoding three copies of the target sites of the (putative) miRNAs indicated above the panels within the 3′ UTR of the CFP gene and which had been mock transfected (Mock) or treated with specific siRNAs to outcompete the relevant miRNA (siRNA). Fitting and statistical analysis were performed as in (B).