MicroRNA Central Loops Figure 5. Effect of loop location on miRNA functioning. The insert fragments of the VEGF 39-UTR in pRL-VEGF-Con1 and/or pRL-VEGF-Con2 were changed to fragments of the c-Met 39-UTR or COX-2 39-UTR to create pRL-CMET-Con3 and pRL-COX2-Con4. These changes allowed us to compare miRNA:MRE duplexes bearing standard central loops (A and D) in miRNA:MRE duplexes with the ones bearing Type I (B) and Type II (E) decentered loops. Luciferase activity assays indicated that changes to Type I and Type II decentered loops decreased the repressive effect of miRNAs significantly (C and F). doi:10.1371/journal.pone.0001719.g005 important rules governing miRNA:MRE interaction were not included. Our results suggest that the location of central loop may be one of the important factors that have not been included. It is well known that binding of miRNAs with their targets is strongly affected by the nucleotides in the 59 end of the miRNA:MRE duplex[53,59], and that, in contrast to the strict requirement for base pairing at the proximal region, nucleotide mismatch at the distal region seems to be highly tolerated. However, there is hardly any previous discussion about the role of the loop region of the miRNA:MRE duplex on miRNA function, although Kiriakidou and coworkers have addressed the effect of the loop size on the efficiency of miRNA-mediated gene regulation[32]. In this investigation, we found that the location of the loop in the miRNA:mRNA duplexes is an important factor which affects the efficiency of gene regulation mediated by miRNAs. Loop scores combining both the size and location of the loops may be used as a new criterion for predicting MREs and their cognate miRNAs, resulting in a significant decrease in false positives. The microarray data from Lim et al. and Grimson et al. provide strong support for the importance of the central loop rule[55,56]. This can be shown through a mathematics approach. The number of predicted MREs in down-regulated genes is assigned to the TD variable, with loop scores . = 20 as SD. The number of predicted MREs in up-regulated genes is then appointed as TU, with loop scores . = 20 as SU. The ratio of positive MREs in down-regulated genes is named a, and the ratio in up-regulated genes b. Because miRNAs are down-regulative factors of gene expression, a should be bigger than b (a.b). Amongst the true positive MREs, the ratio of MREs with loop scores . = 20 is appointed m, and the false positive rate as n. PLoS ONE | www.plosone.org 7 March 2008 | Volume 3 | Issue 3 | e1719