MicroRNA Central Loops Figure 2. Luciferase activity assay. COS-7 cells were co-transfected with the luciferase reporter vector, which contained either the VEGF 39-UTR fragment nt31-216 (pRL-VEGF-Con1) or nt703-944 (pRL-VEGF-Con2), and an miRNA which has a putative binding site in either pRL-VEGF-Con1 (A) or pRL-VEGF-Con2 (B). Luciferase activity was measured to determine the effect of these miRNAs on luciferase translation. All miRNAs have only one predicted MRE in the corresponding report vector. Repressive ratio = (1–LA value of miRNA/LA value of blank)6100%. LA: luciferase activity. The blank is the sample from cells without transfection, providing a control for protein expression in the absence of regulation. A random sequence (NC), miR- 29b, miR-150, miR-106b, and miR-134 that do not have MREs in the VEGF 39-UTR were used as negative controls. doi:10.1371/journal.pone.0001719.g002 We then investigated the effect of loop location on miRNA functioning using naturally existing sites (Figure 5). We changed the fragments of the VEGF 39-UTR in pRL-VEGF-Con1 or 2 to fragments of c-MET or COX2 39-UTR, resulting in the constructs pRL-CMET-Con3 and pRL-COX2-Con4. Change of the 39- UTR fragments allowed us to compare miRNA:MRE duplexes bearing standard central loops (Figure 5A and D) with those bearing Type I (Figure 5B) or Type II (Figure 5E) decentered loops. COS-7 cell were co-transfected with miRNAs and different report constructs, and the levels of luciferase activity measured. The experiments indicated that miRNA:MRE duplexes with Type I or Type II decentered loops caused by changing the fragments showed significantly lower repressive effects, compared with the miRNA:MRE duplexes with standard loops (Figure 5C and F). The results confirmed the importance of the location of central loops in miRNA:MRE in the repressive activity of miRNAs. The effect of adding loop scores as a screening criterion on MRE prediction Since the central loop of miRNA:MRE appeared to be important for miRNA-mediated gene regulation, addition of a loop score as a criterion for MRE prediction should improve the results. To confirm this, all the single MREs in this investigation and MREs in the results (Figure 1, 3, and 6) of Kiriakidou’s publication[32] were included to re-calculate the secondary PLoS ONE | www.plosone.org 4 March 2008 | Volume 3 | Issue 3 | e1719