Prediction and verification of microRNA targets by MovingTargets, a highly adaptable prediction method

Abstract

Background: MicroRNAs (miRNAs) mediate a form of translational regulation in animals. Hundreds of animal miRNAs have been identified, but only a few of their targets are known. Prediction of miRNA targets for translational regulation is challenging, since the interaction with the target mRNA usually occurs via incomplete and interrupted base pairing. Moreover, the rules that govern such interactions are incompletely defined.

Results: MovingTargets is a software program that allows a researcher to predict a set of miRNA targets that satisfy an adjustable set of biological constraints. We used MovingTargets to identify a high-likelihood set of 83 miRNA targets in Drosophila, all of which adhere to strict biological constraints. We tested and verified 3 of these predictions in cultured cells, including a target for the Drosophila let-7 homolog. In addition, we utilized the flexibility of MovingTargets by relaxing the biological constraints to identify and validate miRNAs targeting tramtrack, a gene also known to be subject to translational control dependent on the RNA binding protein Musashi.

Conclusion: MovingTargets is a flexible tool for the accurate prediction of miRNA targets in Drosophila. MovingTargets can be used to conduct a genome-wide search of miRNA targets using all Drosophila miRNAs and potential targets, or it can be used to conduct a focused search for miRNAs targeting a specific gene. In addition, the values for a set of biological constraints used to define a miRNA target are adjustable, allowing the software to incorporate the rules used to characterize a miRNA target as these rules are experimentally determined and interpreted.

Figure 1

Drosophila let-7 miRNA targets the abrupt 3' UTR. A. Predicted sites of let-7 interaction with the ab 3' UTR. The schematic at top shows the relative positions of the sites as vertical bars in the ab 3' UTR. The predicted pairings are shown below, with the free energies (in kcal/mol) and exact positions in the ab 3' UTR indicated. B. Luciferase expression in transfected S2 cells. Expression from the luciferase/ab mRNA with no added miRNA is shown at left, set to a relative value of 1. The other bars indicate the results of coexpression with let-7 or miR-92b. In this figure and in Figure 2, all values represent the average luciferase expression from at least 5 experiments, and error bars represent standard deviation.

Figure 2

miRNA-dependent regulation of CrebA, Eip74 and ttk. Each panel shows at top the distribution of the miRNA target sites in the CrebA (A), Eip74 (B) and ttk (C) 3' UTRs. For panels A and C, the vertical bars above the line indicate sites for miR-92b, while vertical bars below the line indicate sites for miR-312. Luciferase expression data are presented as in Fig. 1.