The Effect of Central Loops in miRNA:MRE Duplexes on the Efficiency of miRNA-Mediated Gene Regulation Wenbin Ye1,2., Qing Lv1,2., Chung-Kwun Amy Wong3, Sean Hu4, Chao Fu1,2, Zhong Hua2, Guoping Cai2, Guoxi Li4, Burton B. Yang3"*, Yaou Zhang2"* 1 Department of Biological Science and Biotechnology, Tsinghua University, Beijing, People’s Republic of China, 2 Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, People’s Republic of China, 3 Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Canada, 4 Shenzhen Beike Biotechnology Co., Ltd., Shenzhen, People’s Republic of China Abstract MicroRNAs (miRNAs) guide posttranscriptional repression of mRNAs. Hundreds of miRNAs have been identified but the target identification of mammalian mRNAs is still a difficult task due to a poor understanding of the interaction between miRNAs and the miRNA recognizing element (MRE). In recent research, the importance of the 59 end of the miRNA:MRE duplex has been emphasized and the effect of the tail region addressed, but the role of the central loop has largely remained unexplored. Here we examined the effect of the loop region in miRNA:MRE duplexes and found that the location of the central loop is one of the important factors affecting the efficiency of gene regulation mediated by miRNAs. It was further determined that the addition of a loop score combining both location and size as a new criterion for predicting MREs and their cognate miRNAs significantly decreased the false positive rates and increased the specificity of MRE prediction. Citation: Ye W, Lv Q, Wong C-KA, Hu S, Fu C, et al (2008) The Effect of Central Loops in miRNA:MRE Duplexes on the Efficiency of miRNA-Mediated Gene Regulation. PLoS ONE 3(3): e1719. doi:10.1371/journal.pone.0001719 Editor: Mark Isalan, Center for Genomic Regulation, Spain Received December 5, 2007; Accepted January 28, 2008; Published March 5, 2008 Copyright: 2008 Ye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a China-Canada Joint Health Research Grant (from Canadian Institutes of Health Research and the National Natural Science Foundation of China to Burton B. Yang and Yaou Zhang), Grant MOP-62729 from Canadian Institutes of Health Research to Burton B. Yang, Grant 30611120519 from the National Natural Science Foundation of China to Yaou Zhang, and Grant 387 2005-k2-046 from the Shenzhen Bureau of Science Technology & Information to Yaou Zhang. Burton B. Yang is the recipient of a Career Investigator Award (CI 5958) from the Heart and Stroke Foundation of Ontario. Competing Interests: The authors have declared that no competing interests exist. * E-mail: byang@sri.utoronto.ca (BBY); zhangyo@sz.tsinghua.edu.cn (YZ) . These authors contributed equally to this work. " These authors are joint senior authors on this work. Introduction MicroRNAs (miRNAs) are small non-coding RNAs which guide translational repression[1–4] or RNA cleavage[5,6] by binding partially or with perfect complementarity with their target sites in the 39-untranslated region (39-UTR) of mRNAs. Having been studied for about a decade, miRNAs have been demonstrated to have important roles in development[7,8], cell proliferation [9– 11], differentiation [12–16], apoptosis[17,18], cell cycle progres- sion [19–21], tumorigenesis[22–26] and many other physiological or pathological processes[2,16,27]. Hundreds of miRNAs have been identified by experimental or bioinformatics methods in animals, plants, and viruses, and the number of miRNAs are still increasing[28]. However, the comprehension of miRNA function remains preliminary, because few miRNA targets have been identified in spite of the enormous quantity of identified miRNAs[5,29,30]. It therefore appears that progress in our understanding of miRNA function has been limited by the difficulty in accurate target prediction and validation. Identification of miRNA targets or the miRNA recognizing element (MRE) is the first step towards understanding the biological function of miRNAs. Since experiments used for the identification of miRNA targets are laborious and are hard to apply for large-scale investigations, many computational approaches have been recently developed to predict miRNA targets in vertebrates and Drosophila[31–38]. However, computa- tional algorithms predicting miRNA targets still have high false- positive rates, because of the poor understanding of the interaction between miRNAs and their cognate mRNAs. Generally, miR- NA:MRE duplexes consist of a 59 end seed region, central bulges or loops (loop region or mismatches), and a 39 end tail region. The importance of the seed region in miRNA-mediated gene regulation is highly emphasized by most researchers, while the effect of the tail region has been addressed computationally by Enright et al. and John et al., and also experimentally by Vella et al., as well as others [34,37,39]. However, the role of the loops in the middle region of the miRNA:MRE duplex is not very clear, though Kiriakidou and coworkers have reported that the size of the loop or bulge might affect the efficiency of miRNA-mediated gene regulation[32]. In this study, we used vascular endothelial growth factor (VEGF) as a model to examine the effect of the loop region in miRNA:MRE duplexes on miRNA-mediated gene regulation, and found that the location of the central loop is another one of the important factors in miRNA functioning. VEGF is an ideal model because the numbers of miRNA binding sites and the various types of miRNA:MRE structures were predicted in the VEGF 39-UTR in our previous investigation[40]. The addition of loop scores PLoS ONE | www.plosone.org 1 March 2008 | Volume 3 | Issue 3 | e1719