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.

Figure 1.

GY-boxes (green circles), Brd-boxes (blue squares), and K-boxes (red triangles) in the 3′-UTRs of Notch target genes of the Brd-Complex and the E(spl)-Complex. These two gene complexes contain 10 members of the Bearded family (A) and seven members of the bHLH repressor family (B). Each 3′-UTR schematic begins with the stop codon and continues to the transcript end as defined by cDNAs and/or consensus polyadenylation signals. Sites are mapped onto D. melanogaster (M) sequences, and those that are conserved in both D. pseudoobscura (P) and D. virilis (V) are dark-filled. Sites that are conserved in only two species are lightly filled, and nonconserved sites are unfilled; species containing these sites are denoted with M, P, or V. The K-box in Brd is starred because it overlaps the stop codon (and thus includes coding sequence), and is present in Dm Brd cDNA but not in Dm Brd genomic DNA (Leviten et al. 1997). The nonconserved Brd-box in E(spl)mδ is in parentheses because it is located ∼10 nt downstream of a utilized polyadenylation site and thus may not formally be located on a 3′-UTR. Note also that there are a limited number of noncanonical box sites in these 3′-UTRs that are not designated in this figure, some of which appear to be functional miRNA-binding sites (see Fig. 6). (C) 3′-UTRs of E(spl)m4 orthologs from the mosquito Anopheles gambiae (Ag), the honeybee Apis mellifera (Am), and the silkmoth Bombyx mori (Bm) also contain multiple GY-boxes, Brd-boxes, and K-boxes.

Figure 2.

Alignment of the 3′-UTRs of a bHLH repressor gene [E(spl)m5 (m5)] (A) and a Bearded family gene (Bearded, Brd) (B) from D. melanogaster (Dm) and D. pseudoobscura (Dp). Conserved regions of three or more nucleotides are marked. Polyadenylation sites from cDNAs are labeled with triangles, and polyadenylation signals are labeled pA; note that E(spl)m5 appears to use a noncanonical signal. GY-boxes, Brd-boxes, and K-boxes are all well-conserved between these species but display two classes of divergence: those that preserve the entire miRNA-binding site (box + upstream sequence) and those that preserve only miRNA seed-pairing (i.e., only the box); certain miRNA-binding sites are also overlapping. Note that the sequence surrounding a nonconserved Brd-box in Dp E(spl)m5 is highly related to the sequence surrounding the conserved third Brd-box of E(spl)m5 (cf. nucleotide identities between underlined sequences).

Figure 3.

miR-7 directly regulates GY-box-containing Notch target genes. All discs contain ptc-Gal4>UAS-DsRed-miR-7 and one copy of tub>GFP (or arm>YFP in the case of Bearded sensors) attached to the 3′-UTRs as designated above each panel pair. The left panel of each pair depicts GFP expression, and the right panel shows the GFP + DsRed merge; miRNA regulation is inferred by diminution of GFP expression in the DsRed domain. Predicted miR-7:GY-box duplexes are shown below each pair, with the miR-7 seed:GY-box pairing highlighted in green; pairings of two or more adjacent nucleotides are marked. Note that the levels of each sensor are not directly comparable as they have been exposed for varying lengths of time.

Figure 4.

miR-4 and miR-79 directly regulate Brd-box-containing Notch target genes. These discs contain dpp-Gal4>UAS-DsRed-miR-4, dpp-Gal4>UAS-DsRed-miR-5, dpp-Gal4>UAS-DsRed-miR-286 (miR-4) (A–J), or dpp-Gal4>UAS-DsRed-miR-79 (K–N) and one copy of tub>GFP (or arm>YFP in the case of Bearded sensors) attached to the 3′-UTRs designated above each panel pair. Predicted miR4/79:Brd-box duplexes are shown below each pair, with the miR-4/79 seed:Brd-box pairing highlighted in blue; pairings of two or more adjacent nucleotides are marked.

Figure 5.

miR-2 and miR-11 directly regulate K-box-containing Notch target genes. These discs contain dpp-Gal4>UAS-DsRed-miR-2a-1, dpp-Gal4>UAS-DsRed-miR-2a-2, dpp-Gal4> UAS-DsRed-miR-2b-2 (miR-2) (A,B) or dpp-Gal4>UAS-DsRed-miR-11 (C–F) and one copy of tub>GFP attached to the 3′-UTRs designated above each panel pair. Predicted miR2/11:K-box duplexes are shown below each pair, with the miR-2/11 seed:K-box pairing highlighted in red; pairings of two or more adjacent nucleotides are marked.

Figure 6.

Exceptional noncanonical GY-box- and Brd-box-like sites are functional. (A) miR-7 regulates the E(spl)mδ 3′-UTR via a GY-box-like site with G:U seed-pairing (asterisk). (B,C) miR-4 and miR-79 both regulate the Bob 3′-UTR via Brd-box-like sites that match positions 2–7 of the canonical Brd-box (variant first positions are marked with asterisks).

Figure 7.

Isolated, endogenous GY-boxes, Brd-boxes, and K-boxes are sufficient and necessary for miRNA-mediated regulation. These discs contain ptc-Gal4>UAS-DsRed-miR-7 (A,B), dpp-Gal4>UAS-DsRed-miR-79 (C,D), dpp-Gal4>UAS-DsRed-miR-4, miR-5, miR-286 (E,F), dpp-Gal4>UAS-DsRed-miR-11 (G), along with tub>GFP sensors containing two copies of GY-box, Brd-box, and K-box sites derived from Bob, Bearded, and E(spl)m8, respectively. Note that the GY-box sensor contains both GY-boxes of Bob (Fig. 3J); miRNA pairing is shown only for the more complementary site. (H,J) Expression of a miR-6 sensor in dpp-Gal4>UAS-miR-6-1, dpp-Gal4>UAS-miR-6-2, dpp-Gal4>UAS-miR-6-3 (miR-6) (H), dpp-Gal4>UAS-miR-2a-1, dpp-Gal4>UAS-miR-2a-2, dpp-Gal4>UAS-miR-2b-2 (miR-2) (I), and dpp-Gal4>UAS-DsRed-miR-11 (mir-11) (J) backgrounds.

Figure 8.

Misexpression of Notch target-regulating miRNAs phenocopies many defects associated with loss of Notch signaling. (A) Wild-type adult wing. (B) dpp-Gal4>UAS-miR-7 shows a notched wing and a reduced L3–L4 intervein region. (C) dpp-Gal4>UAS-miR-2a-1, dpp-Gal4> UAS-miR-2a-2, dpp-Gal4>UAS-miR-2b-2 + UAS-miR-6-1, UAS-miR-6-2, UAS-miR-6-3 displays a notched wing, loss of anterior crossvein, and occasional L3 vein breaks. (D) bx-Gal4>UAS-miR-7 wing shows extensive vein thickening. (E) Cut (green) expression at the developing wing margin of a third instar wing imaginal disc. (F) ptc-Gal4>UAS-DsRed-miR-7 disc displays loss of Cut staining (arrowhead) in the miR-7-expressing domain (red). (G) dpp-Gal4> 2×UAS-DsRed-miR-2a-1, dpp-Gal4>2×UAS-DsRed-miR-2a-2, dpp-Gal4>2×UAS-DsRed-miR-2b-2 shows loss of Cut staining in the miR-2-expressing domain (arrowhead). (H) Wing margin expression of Cut is maintained in a dpp-Gal4>2×UAS-DsRed-miR-79 disc. (I) Wild-type adult notum displays a characteristic pattern of microchaetes (small bristles) and macrochaetes (large bristles); (DC) dorsocentral region; (SC) scutellar region. (J) bx-Gal4-UAS-miR-6-1, bx-Gal4-UAS-miR-6-2, bx-Gal4-UAS-miR-6-3 displays a strong increase in microchaete density and ectopic dorsocentral macrochaetes. (K) bx-Gal4>UAS-miR-2a-1, bx-Gal4-UAS-miR-2a-2, bx-Gal4-UAS-miR-2b-2 shows ectopic dorsocentral and scutellar macrochaetes. (L) Wild-type sternopleural bristles. (M) dpp-Gal4>UAS-miR-7 shows tufted sternopleural bristles. (N) Senseless staining of a wild-type imaginal disc highlights sensory organs precursor (SOP) cells; arrow points to SOPs in the dorsal radius. (O) dpp-Gal4>2×UAS-DsRed-miR-7 shows a strong increase in SOPs in the dorsal radius; asterisk denotes a break in the wing margin.