Table 3. Prediction of true and false MER with FindTar Down-regulated Genes Up-regulated Genes True False True False Positive MER Positive MER Positive MER Positive MER FindTar ver 1.0 aTD FindTar ver 2.0 amTD (1-a)TD (1-a)nTD bTU bmTU (1-b)TU (1-b)nTU doi:10.1371/journal.pone.0001719.t003 Evidence has shown that Ago, the major component of RISCs, includes both PAZ and PIWI domains. The PIWI domain of Ago, containing the triad of acidic amino acids DDH, is responsible for mRNA cleavage in RISCs, in which mRNA cleavage occurs between residues base-paired to nucleotides 10 and 11 of the siRNA, which is equivalent to the location of the standard loop of miRNA:mRNA duplexes. In contrast to the mRNA-cleaving RISC however, little is known about miRNPs or miRISCs. However, the position of the miRNA within the mature complex must be similar to that of the siRNA guide strand, because miRNPs can cleave mRNA when presented with highly complementary targets [60]. What factors decide whether mRNA Table 4. Comparison of repressive ratio. Standard Loop miR-15a/VEGF-Con2 microRNA/mRNA Loop Score Energy Tail Score Seed nts 0.0002 0.0001 0.1738 0.0794 0.7702 MicroRNA Central Loops will be cleaved or translationally repressed when bound by miRNP or an siRNA RISC is not clear, but we believe that the loop of the miRNA:mRNA duplex may be one of the important factors. One element is the location of the standard loop. It is known that perfect complementarity in the central part of the siRNA:mRNA duplexes allows A-form helix formation in the region facing the DDH triad, which is mandatory for cleavage [60]. The presence of a loop in this region effectively interferes with A-helix formation and would probably prevent cleavage. The other reason for the importance of a central loop in switching miRNPs or miRISCs from cleavage of the target to translational repression, is that the loop of the miRNA:mRNA duplex might affect the conformation of Ago. When siRNA or miRNA interact with Ago, its 59 end binds to PIWI and the 39 end to the PAZ domain of Ago. If there is flexible loop between the two ends, it would be easy for Ago to change its conformation to a new one which is necessary for translational repression. Recently released data demonstrates that there are very complex interactions between miRNAs, mRNAs, miRNP pro- teins, and polyribosomes. It is reported that miRNP proteins, including Ago, are physically associated with the polyribosome, and that most miRNAs are also associated with polysomes. Association of miRNAs with polysomes is mRNA-mediated, with Structure GUGUUUGGUAAUACACGACGAU :*||::*|||****|||||:** TTCAGGACAT----TGCTGTGC GCGG--------UUAUAAAUGCACGACGAU *||:******************||||||** TGCTTTGGGGATTCCCTCCACATGCTGCAC UGCGAGUUUACAGCG-------UCGUGAAA ||||*||**|*||||*******:|||||** ACGCGCA--TCTCGCCCCCAGGGGCACTGC GAUGGUAUCCCAUUUUGGUGA *************|||||||* ACGATCGATACAGAAACCACG GGGAGACCAGUU--GGUCAGUGU *||*|||*||::***|||||||* TCC-CTGATCGGTGACAGTCACT TGCGAGTTTAGAGCG-------ACGTGATA ||||****|||||||********|||||:* ACGC--GCATCTCGCCCCCAGGGGCACTGC UGAUG-GACGUGUCUACAGUGAAAC ***||*||||:||||***||||||| GAGACTCTGCGCAGA--GCACTTTG AGAGACGUCCGGCACAC--------GAAACG ***|||||||||*||*|********:||||| GCACTGCAGGCC-TG-GTACTCAGATTTTGC UAUCUCACGUCUGGUCCCAGA *******|||****||||||| TATATCAGCA--AAAGGGTCT UGCGUCUCGGGCUUUCG---GGGGUCA **|||*||:::**|:|:***||:|||* TTGCA-AGTTTTCAGGTAAACCTCAGC UGUGUCCUGGACCU---CAGUCCUC :|||***:||||||****||||||| March 2008 | Volume 3 | Issue 3 | e1719 215.6 222.7 227.6 215.7 223.6 227.5 229.5 227.4 219 220.6 229 9 11 10 9 10 27 27 37 27 9 16 27 6 6 6 7 7 6 7 6 7 6 7 miR-16/VEGF-Con2 miR-372/VEGF-Con2 miR-140/VEGF-Con1 miR-134/VEGF miR-330/Cox2-Con4 miR-504/Cox2-Con4 miR-637/Cox2-Con4 miR-378/VEGF-Con2 210.41 IR% 34.05 40.65 50.18 51.85 45.06 11.80 9.03 16.58 15.86 20 25 20 25 25 10 0 10 10 10 Type I Loop miR-372-M1/VEGF-Con2 miR-17-5p-M1/VEGF-Con1 -12.45 12.5 p-value *Statistics analysis by Student’s test. doi:10.1371/journal.pone.0001719.t004 PLoS ONE | www.plosone.org