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microRNAs (miRNAs) are ~21–22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. We explored this concept in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensively in vivo phenotypic assays, we identify an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. We utilize secondary assays to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, we modeled this situation by asking whether activation of anti-apoptotic miRNAs could serve as “second hits”. Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, we find that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of our screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings.

MicroRNAs (miRNAs) are critical post-transcriptional regulators that may collectively control a majority of animal genes. With thousands of miRNAs identified, a pressing challenge is now to understand their specific biological activities. Many predicted miRNA:target interactions only subtly alter gene activity. It has consequently not been trivial to deduce how miRNAs are relevant to phenotype, and by extension, relevant to disease. We note that the major signal transduction cascades that control animal development are highly dose-sensitive and frequently altered in human disorders. On this basis, we hypothesize that developmental cell signaling pathways represent prime candidates for mediating some of the major phenotypic consequences of miRNA deregulation, especially under gain-of-function conditions. This perspective reviews the evidence for miRNA targeting of the major signaling pathways, and discusses its implications for how aberrant miRNA activity might underlie human disease and cancer.

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.

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology., Author Summary microRNAs are abundant ∼22 nucleotide RNAs inferred to mediate pervasive post-transcriptional control of most genes. Still, relatively little is understood about their endogenous requirements and impact, especially in animal systems. We analyzed a knockout of Drosophila mir-124, which is conserved in sequence and neuronal expression across the animal kingdom, and predicted to have hundreds of mRNA targets. While dispensable for gross neural specification and differentiation, deletion of mir-124 caused short lifespan, increased variation in dendrite numbers, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Loss of miR-124 broadly upregulated its direct targets but did not support the proposed mutual exclusion model, as its functional target genes were relatively highly expressed in neurons. One notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons phenocopies loss of miR-124. Derepression of the direct miR-124 target network had many secondary effects, including over-activity of other post-transcriptional repressors and impaired transition from neuroblast to neuronal transcriptome signatures. Altogether, we demonstrate complex requirements for this conserved miRNA on gene expression and neurophysiology.

The existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA–target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum , which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA–target relationships because other miRNAs with conserved sites in the Axin 3′ UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences.

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.