Despite rapid progress in discovering miRNA genes in organisms as diverse as Caenorhabditis elegans and humans, miRNA target-finding has proved more challenging. Lai et al. now show that the Notch signalling pathway is a major target of miRNA-mediated regulation in Drosophila melanogaster and provide further insights into miRNA target recognition.

The Notch pathway is a signalling cascade that is is essential for cell specification and development in all metazoan organisms. In D. melanogaster, two large families of Notch target genes are clustered at two genomic locations, namely the Enhancer of split-Complex (E(spl)-C) and the Bearded-Complex (Brd-C). Previous genetic and informatics work defined multiple classes of negative regulatory sequence motif that are broadly distributed in the 3′ UTRs of these Notch target genes. These motifs, known as the GY box, the Brd box, and the K box, function to restrict Notch target gene activity during normal development and patterning of the fly nervous system. The authors noticed that the three classes of 'box' motif are perfectly complementary to the 5′ ends of three different families of D. melanogaster miRNAs. Taken together, these observations strongly indicate that all of these boxes are in fact binding sites for miRNAs.

Using an in vivo assay in D. melanogaster imaginal discs to systematically test Notch target 3′ UTRs for regulation by miRNAs, they showed that reporter constructs linked to GY-box-containing 3′ UTRs are inhibited by miR-7, those with Brd boxes by miR-4 and miR-79, and those with K boxes by miR-2 and miR-11. Mutation of the boxes within these 3′ UTRs abolished regulation in this assay, demonstrating that these miRNAs directly target these sites. Other tests using isolated sites further documented the sufficiency and necessity of GY-box, Brd-box and K-box motifs for regulation by complementary miRNAs. This study therefore complements other computational and tissue culture studies with comprehensive in vivo proof that Watson–Crick pairing between a 6–7 nucleotide 3′-UTR box and the 5′ end of the miRNA is a characteristic feature of miRNA-mediated regulation.

So what happens when miRNAs are ectopically expressed during D. melanogaster development? Although computational studies certainly have predicted these Notch target-regulating miRNAs to also target other transcripts that are unrelated to Notch signalling, their misexpression induced several developmental defects that are characteristic of Notch pathway inhibition. This indicates that these Notch pathway target genes are indeed key endogenous targets of these miRNAs.

As the authors note, future studies will need to address how different miRNA-binding sites collectively contribute to the overall regulation of an individual gene, as most of these Notch target genes contain functional binding sites for two or more different miRNAs. Whether miRNA-mediated regulation is a conserved feature of Notch target genes in other animals remains to be determined.