Both miRNA and miRNA* species can be detected in total RNA. RNA was analyzed from D. melanogaster cells at different stages: E, 0–24 h embryos; LP, 3rd instar larvae and mixed pupae; A, adult males and females; S, S2 cells. Each blot was sequentially hybridized, stripped and rehybridized to detect a miRNA* species, its partner miRNA species, then 2S rRNA (the exception is miR-10*, whose companion 2S blot is shown in Supplementary Fig. 1). Arrows indicate the mature 21–24 species and brackets indicate the pre-miRNA hairpin.

miRNA and miRNA* species can be co-immunoprecipitated with AGO1. Each blot contains input total RNA, the immunoprecipitate (IP) of mouse anti-T7 (as a control) and mouse anti-AGO1, and the supernatant (sup) of anti-T7 and anti-AGO1 incubations. IPs were performed from S2 cells and 0–10-h-old embryos, as indicated, and probed for the indicated small RNAs. In the cases of mir-34, mir-5 and mir-10, both miRNA and miRNA* were effectively coimmunoprecipitated with AGO1 but not T7 antibody; miR-184* and miR-276a* were weakly, but specifically, coimmunoprecipitated with AGO1.

Bioinformatic evidence for the endogenous usage of both miRNAs and miRNA* species as regulatory RNAs. (a) miRNA-miRNA* sequence evolution. Above is a schematic of a typical miRNA hairpin, showing that the miRNA seed pairs to the 3′ end of the miRNA*, and vice versa. Analysis of all miRNAs (below, dark green) shows that miRNA termini are more conserved than their central regions, but that windows 1–7 and 2–8 are the most highly conserved. Similar trends apply to the subset of 65 highly conserved (HC) miRNAs and miRNA* species; the highest-scoring portion of the graph has been enlarged (middle). (b) The relative conservation of 3′ UTR complements to 7-nt windows across the 65 HC miRNA-miRNA* (above) and 20 poorly conserved (PC) miRNA/miRNA* (below), assessed between D. melanogaster and its distant relatives D. mojavensis and D. virilis. There is preferential conservation of heptamers complementary to positions 1–7 and 2–8 (and, to a lesser extent, 3–9) of both miRNAs and miRNA* species. (c) The proportion of 3′ UTR matches to miRNA 2–8 seeds that are conserved between D. melanogaster (Dm) and the increasingly divergent species D. simulans (Ds), D. yakuba (Dy), D. ananassae (Da), D. pseudoobscura (Dp), D. mojavensis (Dmo) and D. virilis (Dv). The signal-to-noise ratio (S/N) of HC miRNA and miRNA* seeds, but not of PC seeds, increases steadily with evolutionary distance. (d) Estimated number of conserved target sites for HC miRNAs-miRNA*s. miRNA and miRNA* seeds with similar hit frequencies as their controls in Dm (left) have more conserved matches than their controls in Dmo/Dv (right).

The inherent dual-regulatory capability of miRNA hairpins may influence miRNA evolution. (a) Although bulk miRNA* species are degraded, miRNA hairpins are designed to load specific fractions of miRNA and miRNA* species into AGO complexes. As with miRNA strands, miRNA* functionality has been accompanied by their incorporation into endogenous regulatory networks. (b) The relative ratio of left-arm and right-arm products can differ among members of the same miRNA family. In some cases, the dominant ‘miRNA’ strand has switched between family members. Other miRNA families share a preferred strand, but the level of strand asymmetry can differ dramatically among family members. Perhaps these are miRNA loci in the midst of ‘switching’ their dominant arm.

Highly conserved miRNA* species accumulate to higher relative levels at steady state. (a) Evolution of the mir-309->mir-6 cluster. Both miRNA (green) and miRNA* (yellow) sequences of mir-4 and mir-5 are perfectly conserved across 12 Drosophilids. The other six miRNA* species in this cluster all accumulated divergence (red nucleotides); mir-6-1 is shown. (b) Temporal dynamics of miRNA:miRNA* ratios. Six genes show read ratios that rise progressively with embryo age (time after egg laying (AEL)). Only the genes with highly conserved miRNA* (mir-4 and mir-5) maintain a low miRNA:miRNA* ratio. (c) The miRNA:miRNA* ratios of all 26 miRNA loci that generate at least 50 clones in each of four embryonic time points. Loci whose miRNA* species is perfectly conserved in 11 or 12 Drosophilids are in blue; all other loci are in gray. The most highly conserved miRNA* species tend to be present at a more comparable level to their miRNA partners and are concentrated at the bottoms of these graphs.

Sensor assays in cultured cells and transgenic animals validate the regulatory activity of miRNA* species. (a) Repression by miRNA* species (colored bars) from canonical precursors. Luciferase sensors bearing complementary sites to miR-iab-4-5p/miR-iab-3p and miR-iab-8-5p/miR-iab-8-3p were all repressed by cognate but not noncognate pre-miRNAs. Mean values and s.d. are shown. (b) Repression by miRNA* species from a mirtron precursor. Sensors for both miR-1010 and miR-1010* were repressed by mir-1010 but not by the unrelated mirtron mir-1003. (c,d) Repression of miRNA* targets in transgenic animals. Shown are the wing pouch regions of third instar imaginal discs that express the indicated DsRed-miRNA constructs and GFP sensors. Inhibition of both 5p and 3p sensors by mir-iab-4 (c) and mir-iab-8 (d) is reflected by the loss of GFP in DsRed⁺ cells.

Endogenous relevance of miRNA*-mediated repression. (a) Ectopic mir-276a and mir-276b, but not DsRed or mir-315, specifically repress both miR-276a-5p and miR-276a-3p sensors. The experimental design is the same as for Figure 4a; mean values and s.d. are shown. (b) 2′O-methylated antisense oligos (ASO) against endogenous miR-276a-5p and miR-276a-3p specifically derepress cognate sensors. Data were collected and analyzed as in a. (c) Abrupt is an endogenous target of the miRNA* species, miR-iab-4-3p. Of three ‘2–8’ seed matches (highlighted yellow) near the start of the abrupt 3′ UTRs (denoted ab#1, ab#2 and ab#3) two are highly conserved among divergent Drosophilids and one has a t1A feature (red). (d) Endogenous Abrupt protein in a wing pouch carrying one copy of ptc-Gal4 and two copies of UAS-DsRed. Abrupt accumulates to a high level in the L5 wing vein primordium and a lower level in the L3 vein domain (arrows). (e) Wing pouch of an animal carrying two copies of ptc-Gal4 and one copy of UAS-DsRed-mir-iab-4. Abrupt protein is reduced in L3 (arrows). Genotypes d and e express roughly equivalent amounts of DsRed and control for the neutral effect of DsRed on Abrupt. (f) In ptc-Gal4, UAS-DsRed-mir-iab-4; tub-GFP-abrupt 3′ UTR wing imaginal discs, GFP is strongly suppressed in DsRed/miR+ cells.