The recent discoveries of microRNAs (miRNAs) and characterization of the first few targets of their gene products in Caenorhabditis elegans and Drosophila melanogaster have set the stage for elucidation of a novel network of regulatory control. Here, we present a novel three-step method for whole-genome prediction of miRNA target genes, validated using known examples. We apply the method to discover hundreds of potential target genes in D. melanogaster. For each miRNA, target genes are selected based on (a) pattern of sequence complementarity using a position-weighted local alignment algorithm, (b) energy calculation of RNA-RNA duplex formation, and (c) conservation of target sites in related genomes. Application to the D. melanogaster, D pseudoobscura and Anopheles gambiae genomes in this manner, identifies several hundred target genes potentially regulated by one or more known miRNAs.

These potential targets are enriched for genes that are expressed at specific developmental stages and are involved in cell fate specification, morphogenesis and the coordination of developmental processes, as well as the function of the nervous system in the mature organism. High-ranking targets are two-fold enriched in transcription factors and include genes already known to be under translational regulation. Our results reaffirm the thesis that miRNAs play an important role in establishing the complex spatial and temporal patterns of gene activity necessary for the orderly progression of development and point to additional roles in the function of the mature organism.

The emerging combinatorics of miRNA target sites in the 3' UTRs of messenger RNAs are reminiscent of transcriptional regulation in promoter regions of DNA, with both one-to-many and many-to-one relationships between regulator and regulated target. Typically, more than one miRNA regulates one message, indicative of cooperative control of translation. Conversely, one miRNAs may have several targets, reflecting target multiplicity.

As a guide to targeted experiments, we provide detailed online information [1] about target genes and binding sites for each miRNA and about miRNAs for each gene, ranked by likelihood of match. The target prediction tool can be applied to any similar pair of genomes with identified miRNA sequences.