Misexpression of miR-iab-4AS transforms halteres to wings. (A,B) Overview of an adult wild-type Drosophila (B) and an adult expressing mir-iab-4AS using bx-Gal4 (A). The halteres, balancing organs of the third thoracic segment, are indicated by arrows. (C) Wild-type haltere. (D) Expression of mir-iab-4 using bx-Gal4 induces a mild haltere-to-wing transformation. Sensory bristles characteristic of wild-type wing margins (shown in B′) are indicated by an arrow. (E) Expression of mir-iab-4AS using bx-Gal4 induces a strong haltere-to-wing transformation, displaying the triple row of sensory bristles (inset) normally seen in wild-type wings (shown in B′). Note that C–E are at the same magnification.

miR-iab-4AS targets neighboring Hox genes. (A) miR-iab-4AS has five 3′ UTR seed sites (red) in Ubx, four in abd-A, and two in Antp of which three, four, and one are conserved across 12 Drosophila species, respectively (Supplemental Fig. S2). miR-iab-4 has one 3′ UTR seed site (blue) in Ubx and two in Antp, while abd-A has no such sites. (B) miR-iab-4AS mediates repression of luciferase reporters through complementary seed sites in 3′ UTRs from abd-A and Ubx, but not Abd-B (Antp was not tested). Luciferase activity in S2 cells cotransfected with plasmid expressing the indicated miRNA with either wild-type luciferase reporters or mutant reporters bearing a single point mutation in the seed. Bars represent geometric means from 16 replicates, normalized to the transfection control and noncognate miRNA control (let-7; see Materials and Methods). Error bars represent the fourth largest and smallest values from 16 replicates ([*] P < 0.0001, Wilcoxon rank-sum test).

Drosophila iab-4 contains sense and antisense miRNAs. (A) mir-iab-4 sense and antisense sequences can adopt fold-back stem–loop structures characteristic for miRNA precursors (structure predictions by Mfold [Zuker 2003]; mature miRNAs shaded in blue [miR-iab-4] and red [miR-iab-4AS]). (B) Solexa sequencing reads that uniquely align to the mir-iab-4 hairpin sequence (top) or its reverse complement (bottom; numbers on the right indicate the cloning frequency for each sequence). The mature miRNAs have very similar sequences that are shifted by 2 nt and are different in only four additional positions. (C) Expression of primary transcripts for mir-iab-4 (blue) and mir-iab-4AS (red) in nonoverlapping abdominal segments determined by in situ hybridization (lateral [left panel] and dorsal [right panel] view of embryonic stage 11, anterior is to the left). (D) Lateral views of stage 10/11 embryos in which Ubx and abd-A proteins are visualized (anterior is to the left, and dorsal is upwards).

Regulation of gene expression by antisense miRNAs. (A) miRNA-mediated control in the Drosophila Hox cluster. Schematic representation of the Drosophila Hox cluster (Antennapedia and Bithorax complex) with miRNA target interactions (check marks represent experimentally validated targets). miR-iab-4 (blue) and miR-iab-4AS (red) target anterior neighboring Hox genes and miR-10 (black) targets posterior Sex-combs-reduced (Scr) (Brennecke et al. 2005). abd-A and mir-iab-4 and Abd-B and mir-iab-4AS might be coregulated from shared control elements (cis). Note that mir-iab-4AS is expressed in the same direction as most other Hox genes and its mammalian equivalent, mir-196. (B) General model for defining different expression domains with pairs of antisense miRNAs (black). Different transcription factor(s) activate the transcription of miRNAs and genes in each of the two domains separately (green lines). Both miRNAs might inhibit each other by transcriptional interference or post-transcriptionally (vertical red lines), leading to essentially nonoverlapping expression and activity of both miRNAs. Further, both miRNAs likely target distinct sets of genes (diagonal red lines), potentially re-enforcing the difference between the two expression domains.