Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs

Abstract

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3 -untranslated regions (3 -UTRs) that are complementary to the 5 -ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.[Keywords: microRNA; Notch signaling; Enhancer of split-Complex; Bearded-Complex] Supplemental material is available at http://www.genesdev.org.

Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development

“…4 G-I and data not shown). These phenotypes are identical to those conferred by a UAS-miR-7 transgene, consistent with the finding that seven Notch targets are directly repressed by miR-7 (19,27). In contrast, misexpression of (28).…”

“…The observation that one of the vertebrate miR-7 genes similarly resides in an intron of hnRNP-K suggested an intimate functional association between them. However, in vivo analyses demonstrate that key miR-7 targets include Hairy͞E(spl)bHLH repressor genes and Bearded family genes (19,27). These genes function in segmentation, sensory organ development, and Notch signal transduction and are not ostensibly linked to hnRNP-K function.…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…In fact, it is well established that the perfect complementary between the miRNA 2-8 nts, so called 'seed sequence', and the targeted RNA is absolutely essential for target recognition. In this region, a single mismatch blocks binding, although a shorter seed pairing may be compensated by extensive matching between the target and the 3 0 regions of the miRNAs (Lewis et al, 2003;Kloosterman et al, 2004;Brennecke et al, 2005;Lai et al, 2005). Another function of the 3 0 region of a miRNA is to give specificity to the members of miRNA families that share identical seeds but they diverge in their 3 0 sequences (Brennecke et al, 2005).…”

Principles of micro-RNA production and maturation

“…7 positions (No. 1 being overrepresented by a U), are critical for target recognition (Doench and Sharp, 2004;Mallory et al, 2004;Brennecke et al, 2005;Krek et al, 2005;Lai et al, 2005;Lewis et al, 2005). Prediction of miRNAs by current computational methods or detection of miRNAs by Northern blot and microarray analyses cannot pinpoint the ends of miRNAs.…”