BMC Genomics 2005, 6:88 http://www.biomedcentral.com/1471-2164/6/88 with the miRNA target database sequences, producing a set of predicted targets. The user sets values for the con- straints. This adjustable algorithm facilitates focused searches with individual mRNAs, where experimental evi- dence may suggest that miRNA-dependent regulation exists. 1) Number of target sites in the mRNA. For most of the known miRNA/target mRNA pairs the miRNA is predicted or known to interact with multiple sites within the 3' UTR [23-28]. Furthermore, there is experimental evidence of a synergistic effect between multiple miRNP complexes associated with a single mRNA [21], suggesting that mul- tiple target sites may allow for rapid translational control [32]. 2) Strength of miRNA-mRNA hybridization. The specific interaction of a miRNA with a target mRNA involves base pairing [4], and it is reasonable to assume that target site occupancy will be positively correlated with the strength of the base pairing [22-28]. We therefore rank potential miRNA/target interactions according to the strength of hybridization between the miRNA and its target site, as measured by the predicted free energy of binding. These predictions are made using M. Zuker's DINAMelt Server software [33] which was expressly designed for evaluating the interactions of short RNAs and thus offers advantages over the commonly used alternatives, mFold [34] and RNAfold [35]. 3) Number of consecutive base pairs involving the 5' part of the miRNA. There is suggestive evidence that miRNA/ target interactions require a series of consecutive base pairs between the 5' part of the miRNA and the target [7,23-28,36-38]. Of the experimentally validated animal miRNA targets [2,23-28], 19 of the 24 predicted miRNA/ mRNA interactions have 6 or more consecutive base pairs within the first 8 nucleotides of the miRNA; 10 of these interactions have perfect complementarity in this region. This is contrasted with only 5 of the 24 predicted miRNA/ mRNA interactions having 6 or more consecutive base pairs at the miRNA 3' end. Note that for almost all exam- ples of mRNAs known to be regulated by miRNAs, the specific target sites in the mRNA (identified as regions with significant complementarity to the miRNA) have not been individually tested and verified. Additional evidence comes from mutational analysis of miRNAs and their targets. The ability of miR-30 to repress translation of an artificial target in cultured human cells is eliminated by a mutation in the target mRNA that disrupts a single base pair in the middle of the 5' region of the miRNA, while a mutation in the target mRNA disrupting base pairing in the 3' part of the miRNA retains about 60% of the repressive activity [22]. We used the DINAMelt Server to predict the effect of both mutations on hybridi- zation strength, and found that the inactivating mutation had a more modest effect than the weak mutation. These results argue that the important aspect of the interaction disrupted by the first mutation was the consecutive series of base pairs at the 5' end of the miRNA, rather than the strength of the interaction as measured by thermody- namic stability considerations alone. A mutation in the 5' region of the let-7 miRNA eliminates repression of lin-41 mRNA in vivo, but also reduces the level of the mature miRNA, making it difficult to conclude why it is ineffec- tive [3]. 4) Total number of miRNA 5' nucleotides involved in base pairing to the target. For mRNAs shown to be miRNA tar- gets, all of the 24 predicted miRNA/mRNA interactions have 6 or more total base pairs within the first 8 nucle- otides of the miRNA; 21 of these interactions have at least 7 base pairs. This is contrasted with only 11 of the 24 pre- dicted miRNA/mRNA interactions having 6 or more total base pairs at the miRNA 3' end [23-28]. In addition, there is more stringent sequence conservation in the 5' end of homologous miRNAs than in the 3' end [36]. 5) Number of nucleotides in the miRNA 5' region involved in G:U base pairs. Predicted miRNA/target inter- actions of known miRNA targets have at most one (6 out of the 24 predicted miRNA/mRNA interactions) and usu- ally no G:U base pairs in the miRNA 5' region. In contrast, despite having fewer overall base pairs in the miRNA 3' region, 9 out of the 24 predicted miRNA/target interac- tions have more than 1 G:U base pair in the miRNA 3' region, and 8 of the 24 have 1 G:U base pair in this region [23-28]. Thus, canonical base pairing appears to be favored over G:U base pairing in the miRNA 5' region. Subsequent to development of the MovingTargets algo- rithm an extensive study of the rules of miRNA/target interactions was published [39]. The results emphasize the importance of the latter three constraints described above. Methods DNA constructs Reporter plasmids were constructed by cloning the 3'UTR of each target gene into the BamHI/XbaI site of luk-ttkUTR [40]. luc/tramtrack is the luk-ttkUTR plasmid. For luc/ CrebA, the CrebA 3'UTR was amplified by PCR from genomic DNA (all sequence coordinates are from Release 3 from FlyBase, http://www.flybase.net): 3L:15500154- 15502103. For luc/ab, the ab 3'UTR was PCR amplified from genomic DNA: 2L:11248260-11249979. For luc/ Eip74EF, the Eip74EF 3'UTR beginning at position 29 was cloned from pBSE74AcDNA, a gift from Carl Thummel. Page 3 of 11 (page number not for citation purposes)