The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into approximately 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs.

During microRNA (miRNA) biogenesis, one strand of a ∼21-22-nucleotide RNA duplex is preferentially selected for entry into a silencing complex. The other strand, known as the miRNA* species, has typically been assumed to be a carrier strand. Here we show that, although Drosophila melanogaster miRNA* species are less abundant than their partners, they are often present at physiologically relevant levels and can associate with Argonaute proteins. Comparative genomic analyses revealed that >40% of miRNA* sequences resist nucleotide divergence across Drosophilid evolution, and at least half of these well-conserved miRNA* species select for conserved 3′ untranslated region seed matches well above background noise. Finally, we validated the inhibitory activity of miRNA* species in both cultured cells and transgenic animals. These data broaden the reach of the miRNA regulatory network and suggest an important mechanism that diversifies miRNA function during evolution.miRNAs are an abundant class of ∼21-22-nucleotide (nt) RNAs that typically function as posttranscriptional repressors of gene activity 1,2 . The biogenesis of animal miRNAs involves stepwise processing of precursor transcripts containing hairpin structures. Canonical primary miRNA transcripts are cleaved in the nucleus by the RNase III enzyme Drosha, releasing ∼60-80-nt pre-miRNA hairpins 3 . In addition, splicing and debranching of short hairpin introns termed 'mirtrons' can directly generate pre-miRNA-like hairpins 4-6 . In both cases, the hairpins are exported to the cytoplasm and cleaved by the RNase III enzyme Dicer, resulting in a ∼21-nt miRNA duplex 7-10 . Although both strands of miRNA duplexes are necessarily produced in equal amounts by transcription, their accumulation is asymmetric at steady state. The convention is to refer to the more abundant product of a pre-miRNA or mirtron hairpin as the miRNA and its rarer partner as a miRNA* species 11 .The function of miRNA strands is evident from the preferential conservation of 7-nt sequences in target transcripts with Watson-Crick complementarity to positions 2-8 of mature miRNAs (the 'seed' region). Although other features influence target-site efficacy, miRNA seed matches are often necessary and sufficient for target regulation 12-15 and are the basis of most genomewide predictions of miRNA regulatory sites 16-18 . Such studies conclude that most animal genes are either actively regulated by one or more miRNAs or actively avoid the acquisition © 2008 Nature Publishing Group Correspondence should be addressed to E.C.L. (E-mail: laie@mskcc.org 17,19 . The reach of the miRNA regulatory network may in fact be larger, depending on the extent to which additional miRNA genes remain to be discovered, the extent to which noncanonical target sites are functional, and the extent to which nonconserved sites are relevant in vivo 20 .The nonrandom nature of miRNA strand selection was posited to reflect an active process that minimizes the population of silencing complexes with illegitimate miRNA* speci...

Many microRNA (miRNA) loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. We report here that antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. We find that miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. We generalize the antisense miRNA principle by showing that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes.[Keywords: BX-C; antisense; homeotic gene; microRNA] Supplemental material is available at http://www.genesdev.org.

Cell competition is a homeostatic mechanism that regulates the size attained by growing tissues. We performed an unbiased genetic screen for mutations that permit the survival of cells being competed due to haplo-insufficiency for RpL36. Mutations that protect RpL36 heterozygous clones include the tumor suppressors expanded, hippo, salvador, mats, and warts, which are members of the Warts pathway, the tumor suppressor fat, and a novel tumor-suppressor mutation. Other hyperplastic or neoplastic mutations did not rescue RpL36 heterozygous clones. Most mutations that rescue cell competition elevated Dppsignaling activity, and the Dsmurf mutation that elevates Dpp signaling was also hyperplastic and rescued. Two nonlethal, nonhyperplastic mutations prevent the apoptosis of Minute heterozygous cells and suggest an apoptosis pathway for cell competition . In addition to rescuing RpL36 heterozygous cells, mutations in Warts pathway genes were supercompetitors that could eliminate wild-type cells nearby. The findings show that differences in Warts pathway activity can lead to competition and implicate the Warts pathway, certain other tumor suppressors, and novel cell death components in cell competition, in addition to the Dpp pathway implicated by previous studies. We suggest that cell competition might occur during tumor development in mammals.

Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.

microRNAs (miRNAs) are an abundant class of ~22 nucleotide (nt) regulatory RNAs that are pervasive in higher eukaryotic genomes. In order to fully understand their prominence in genomes, it is necessary to elucidate the molecular mechanisms that can diversify miRNA activities. In this review, we describe some of the many strategies that allow novel miRNA functions to emerge, with particular emphasis on how miRNA genes evolve in animals. These mechanisms include changes in their sequence, processing, or expression pattern; acquisition of miRNA* functionality or antisense processing; and de novo gene birth. The facility and versatility of miRNAs to evolve and change likely underlies how they have become dominant constituents of higher genomes.

Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5-TGGC ACGTG(C/T)(C/T)A-3, which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.The basic helix-loop-helix (bHLH) family of transcription factors includes many members that mediate cell fate allocation during animal development (30,39,45,71). Their expression and activity can be regulated in response to cell-cell signalling, leading to the transcription of the specific set of genes required for a cell to adopt a particular fate. One pathway whose effect on cell fate decisions involves modulation of bHLH proteins is the Notch signalling pathway (reviewed in references 2 and 25). The most immediate transcriptional target genes of Notch activation in Drosophila melanogaster encode seven bHLH proteins (M␦, M␤, M␥, M3, M5 M7, and M8) which are clustered in the Enhancer of split complex [E(spl)-C] (14, 36, 37). A number of closely related genes, known as Hes, Her, or ESR genes (44,55,60,62), have now been isolated from vertebrates, and like the Drosophila E(spl) genes, many of the vertebrate homologues are expressed in response to Notch activity (3,13,32,34,38). The products of these genes are essential to implement many of the cell fate decisions mediated by Notch signalling, such as the selection of cells to become neural precursors (2, 25). Thus, a knowledge of the functional characteristics of the E(spl)bHLH proteins should lead to a greater understanding of how the activation of Notch mediates cell fate decisions via changes in gene transcription.The E(spl) proteins represent a subset of bHLH proteins that also includes the Drosophila proteins Hairy and Deadpan (19). One distinguishing feature of this class of bHLH proteins is the presence of a proline residue in the basic domain. The basic domain confers on bHLH proteins DNA binding specificity (5, 10, 17, 18) for which the canonical target sequence is the E box (5Ј-CANNTG-3Ј). Initially it was postulated that the proline residue found in E(spl)-like bHLH proteins would impede DNA binding ability. Subsequently howev...

The Drosophila Bithorax-Complex (BX-C) Hox cluster contains a bidirectionally-transcribed miRNA locus, and a deletion mutant (∆mir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the central nervous system. ∆mir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, since sterility of ∆mir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in ∆mir females, and substantially rescued by heterozygosity of ∆mir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation, and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.