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Meiotic drivers are a class of selfish genetic elements whose existence is frequently hidden due to concomitant suppressor systems. Accordingly, we know little of their evolutionary breadth and molecular mechanisms. Here, we trace the evolution of the Dox meiotic drive system in Drosophila simulans, which affects male-female balance (sex ratio). Dox emerged via stepwise mobilization and acquisition of multiple D. melanogaster gene segments including from protamine, which mediates compaction of sperm chromatin. Moreover, we reveal novel Dox homologs and massive amplification of Dox superfamily genes on X chromosomes of its closest sisters D. mauritiana and D. sechellia. Emergence of Dox loci is tightly associated with 359-class satellite repeats that flank de novo genomic copies. In concert, we find coordinated diversification of autosomal hairpin RNA-class siRNA loci that target subsets of Dox superfamily genes. Overall, we reveal fierce genetic arms races between meiotic drive factors and siRNA suppressors associated with recent speciation.

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.

Meiotic drivers are a class of selfish genetic elements that are widespread across eukaryotes. As their activities are often detrimental to organismal fitness, opposing regulatory mechanisms are usually required to silence them, to ensure fair segregation during meiosis. Accordingly, the existence of such selfish elements is frequently hidden in genomes, and their molecular functions are little known. Here, we trace evolutionary steps that generated the Dox meiotic drive system in Drosophila simulans (Dsim), which distorts male:female balance (sex-ratio) by depleting male progeny. We show that Dox emerged via stepwise mobilization and acquisition of portions of multiple D. melanogaster genes, notably including from protamine, which replaces histones in haploid sperm and mediates the highly condensed state of sperm chromatin. Moreover, we reveal novel Dox homologs in Dsim and massive, recent, amplification of Dox superfamily genes specifically on X chromosomes of its closest sister species D. mauritiana (Dmau) and D. sechellia (Dsech). The emergence of Dox superfamily genes is tightly associated with 359-bp repeats (in the 1.688 family of satellite repeats) that flank de novo genomic copies. In concert, we find coordinated emergence and diversification of autosomal hairpin RNA-class siRNA loci that target subsets of Dox superfamily genes across simulans clade species. Finally, an independent set of protamine amplifications on the Y chromosome of D. melanogaster indicates that protamine genes are frequent and recurrent players in sex chromosome dynamics. Overall, we reveal fierce genetic arms races between meiotic drive factors and siRNA suppressors associated with recent speciation.

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units., The BEN-solo proteins—including Insensitive (Insv), Elba1 and Elba2—function in both transcriptional repression and chromatin insulation. Here, the authors investigate the role of these proteins in Drosophila embryos, finding that ELBA and Insv function as general insulators and partition active chromatin to ensure proper gene activation in Drosophila.

Somatic mutations in the RNase IIIb domain of DICER1 arise in cancer and disrupt the cleavage of 5' pre-miRNA arms. Here, we characterize an unstudied, recurrent, mutation (S1344L) in the DICER1 RNase IIIa domain in tumors from The Cancer Genome Atlas (TCGA) project and MSK-IMPACT profiling. RNase IIIa/b hotspots are absent from most cancers, but are notably enriched in uterine cancers. Systematic analysis of TCGA small RNA datasets show that DICER1 RNase IIIa-S1344L tumors deplete 5p-miRNAs, analogous to RNase IIIb hotspot samples. Structural and evolutionary coupling analyses reveal constrained proximity of RNase IIIa-S1344 to the RNase IIIb catalytic site, rationalizing why mutation of this site phenocopies known hotspot alterations. Finally, examination of DICER1 hotspot endometrial tumors reveals derepression of specific miRNA target signatures. In summary, comprehensive analyses of DICER1 somatic mutations and small RNA data reveal a mechanistic aspect of pre-miRNA processing that manifests in specific cancer settings., DICER is involved in the processing of miRNAs, where the RNase IIIa and IIIb domains are thought to cut the 3p and 5p hairpin arms, respectively. Here, in endometrial cancer, the authors identify an RNase IIIa mutation, which phenocopies mutations in the RNase IIIb domain.

International audience; The fruitfly Drosophila melanogaster has been extensively used as a genetic model for the maintenance of nervous system's functions. Glial cells are of utmost importance in regulating the neuronal functions in the adult organism and in the progression of neurological pathologies. Through a microRNA-based screen in adult Drosophila glia, we uncovered the essential role of a major glia developmental determinant, repo, in the adult fly. Here, we report that Repo expression is continuously required in adult glia to transcriptionally regulate the highly conserved function of neurotransmitter recycling in both males and females. Transient loss of Repo dramatically shortens fly lifespan, triggers motor deficits, and increases the sensibility to seizures, partly due to the impairment of the glutamate/GABA/glutamine cycle. Our findings highlight the pivotal role of transcriptional regulation of genes involved in the glutamate/GABA/glutamine cycle in glia to control neurotransmitter levels in neurons and their behavioral output. The mechanism identified here in Drosophila exemplifies how adult functions can be modulated at the transcriptional level and suggest an active synchronized regulation of genes involved in the same pathway. The process of neurotransmitter recycling is of essential importance in human epileptic and psychiatric disorders and our findings may thus have important consequences for the understanding of the role that transcriptional regulation of neurotransmitter recycling in astrocytes has in human disease.SIGNIFICANCE STATEMENT Glial cells are an essential support to neurons in adult life and have been involved in a number of neurological disorders. What controls the maintenance and modulation of glial functions in adult life is not fully characterized. Through a miR overexpression screen in adult glia in Drosophila, we identify an essential role in adult glia of repo, which directs glial differentiation during embryonic development. Repo levels modulate, via transcriptional regulation, the ability of glial cells to support neurons in the glutamate/GABA/glutamine cycle. This leads to significant abnormalities in motor behavior as assessed through a novel automated paradigm. Our work points to the importance of transcriptional regulation in adult glia for neurotransmitter recycling, a key process in several human neurological disorders.

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments., Author summary The Exon Junction Complex (EJC) is a conserved multiprotein complex that is deposited ~20–24 nucleotides upstream of exon-exon junctions during mRNA splicing. Although the EJC is well-conserved, many of its overt regulatory requirements differ between species. For example, the mammalian EJC is involved in mRNA surveillance and nonsense mediated decay (NMD), and also suppresses cryptic splicing. On the other hand, the Drosophila EJC does not mediate NMD, and it has multiple roles in promoting splicing of long introns and suboptimal splicing substrates. Here, we unify this by showing that the Drosophila EJC suppresses splicing at hundreds of illegitimate cryptic splice sites, which are presently unannotated in the well-studied Drosophila genome. As in mammals, this role takes advantage of the sequence-independent deposition of the EJC upstream of splice sites, and appears to represent an ancestral function. We expand this concept by showing the necessity of the EJC to prevent resplicing in exonic remnants that inherently regenerate splice sites following canonical splicing. Importantly, cDNA expression constructs evade EJC regulation, and we show that utilization of cDNAs can unintentionally trigger re-splicing into unanticipated products with internal deletions.

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons., Author summary ELAV/Hu factors comprise a conserved family of RNA binding proteins (RBPs), many of which are enriched or restricted to neurons. This study shows that overlapping activities of Drosophila ELAV/Hu RBPs determine global patterns of neural alternative splicing, including of cassette exons and alternative last exon (ALE) isoforms. This is supported by both genetic necessity in double mutant CNS, as well as their sufficiency to drive these mRNA processing changes in a non-neuronal setting. The ability of ELAV/Hu RBPs to induce the usage of distal ALE isoforms connects to their recently described functions in global extension of 3’UTRs in neural isoforms. Evidence is provided that switching to distal alternative last exons and to terminal 3’ UTR extensions are also coordinated in mammalian neurons, and show signatures of direct regulation by ELAV/Hu RBPs.

SummaryThe survival and reproductive success of animals depends on the ability to harmonize their external behaviors with their internal states. For example, females conduct numerous social programs that are distinctive to virgins, compared to post-mated and/or pregnant individuals. In Drosophila, the fact that this post-mating switch is initiated by seminal factors implies that the default state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgin females to execute mated behaviors. To elucidate new components of this post-transcriptional regulatory circuit, we used genomic analysis of mir-iab-4/8 deletion and hth-miRNA binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. While Dsx has mostly been studied during sex-specific differentiation, its activities in neurons are little known. We find that accumulation of Dsx in the CNS is highly complementary to Hth, and downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose-sensitive to developmental dsx function. Strikingly, depletion of Dsx in SAG-1 cells, a highly restricted set of abdominal neurons, abrogates female virgin conducts in favor of mated behavioral programs. Thus, a double negative post-transcriptional pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state.

SUMMARY Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. Here, we use genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 ⫞ Hth ⫞ Dsx) specifies the virgin behavioral state., Graphical abstract, In brief Garaulet et al. use transcriptomic analysis to reveal new downstream elements in a post-transcriptional cascade, via miR-iab-4/8 and Homothorax, that affects patterning of the CNS. This genetic circuit regulates the accumulation of a secondary target (Doublesex), whose level in specific neurons determines the behavior of adult virgin flies.

ELAV/Hu factors are conserved RNA binding proteins that play diverse roles in mRNA processing and regulation. The founding member,DrosophilaElav, was recognized as a vital neural factor 35 years ago. Nevertheless, still little is known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. In ectopic contexts, all three members (Elav, Fne and Rbp9) induce similar and broad changes to cassette exon and alternative last exon (ALE) splicing. Reciprocally, double mutants ofelav/fne, but notelavalone, have opposite effects on both types of mRNA processing events in the larval CNS. Accordingly, whilefnemutants are normal,fneloss strongly enhanceselavmutants with respect to neuronal differentiation. While manipulation ofDrosophilaELAV/Hu factors induces both exon skipping and inclusion, motif analysis indicates their major direct effects are to suppress cassette exon usage. Moreover, we find direct analogies in their roles in global promotion of distal ALE splicing and terminal 3’ UTR extension, since both involve local suppression of proximal polyadenylation signals via ELAV/Hu binding sites downstream of cleavage sites. Finally, we provide evidence for analogous co-implementation of distal ALE and APA lengthening programs in mammalian neurons, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu proteins orchestrate multiple conserved programs of neuronal mRNA processing by suppressing alternative exons and polyadenylation sites.

Accurate splice site selection is critical for fruitful gene expression. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic splice sites (SS), even though majority of these bear weak splicing motifs and appear incompetent. Mechanistically, the EJC directly conceals splicing elements through position-specific recruitment, preventing SS definition. We note that intron removal using strong, canonical SS yields AG|GU signatures at exon-exon junctions. Unexpectedly, we discover that scores of these minimal exon junction sequences are in fact EJC-suppressed 5’ and 3’ recursive SS, and that loss of EJC regulation from such transcripts triggers faulty mRNA resplicing. An important corollary is that intronless cDNA expression constructs from aforementioned targets yield high levels of unanticipated, truncated transcripts generated by resplicing. Consequently, we conclude the EJC has ancestral roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

SummaryHow are diverse regulatory strategies integrated to impose appropriately patterned gene expression that underliein vivophenotypes? Here, we reveal how coordinated miRNA regulation and neural-specific alternative polyadenylation (APA) of a single locus controls complex behaviors. Our entry was the unexpected observation that deletion of Bithorax-Complex (BX-C) miRNAs converts virgin female flies into a subjective post-mated behavioral state, normally induced by seminal proteins following copulation. Strikingly, this behavioral switch is directly attributable to misregulation ofhomothorax(hth). We localize specific CNS abdominal neurons where de-repressed Hth compromises virgin behavior in BX-C miRNA mutants. Moreover, we use genome engineering to demonstrate that precise mutation ofhth3’UTR sites for BX-C miRNAs, or deletion of its neural 3’ UTR extension containing most of these sites, both induce post-mated behaviors in virgins. Thus, facilitation of miRNA-mediated repression by neural APA is required for virgin females to execute behaviors appropriate to their internal state.

Summary: MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos. : Huangfu and colleagues report an unexpected requirement for DICER1 in preventing death-receptor-mediated apoptosis in hESCs. This essential pro-survival role is largely mediated by mir-302-367 and mir-371-373 cluster members that bear the seed sequence “AAGUGC” shared by the ESC-specific cell-cycle-regulating family of miRNAs. This work provides a robust platform for interrogating miRNA function in hESC pluripotency and differentiation. Keywords: human embryonic stem cells (hESCs), human pluripotent stem cells (hPSCs), DICER1, microRNAs (miRNAs), self-renewal, apoptosis, ESCC miRNAs

Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.

Recursive splicing is an unusual form of stepwise mRNA processing involving exons with functional splice donors at their 5′ ends. We use molecular and genetic assays to show three parameters that influence mRNA isoform outcomes during recursive splicing in Drosophila., Intronic ratchet points (RPs) are abundant within long introns in the Drosophila genome and consist of juxtaposed splice acceptor and splice donor (SD) sites. Although they appear to encompass zero-nucleotide exons, we recently clarified that intronic recursive splicing (RS) requires a cryptic exon at the RP (an RS-exon), which is subsequently always skipped and thus absent from mRNA. In addition, Drosophila encodes a smaller set of expressed exons bearing features of RS. Here, we investigate mechanisms that regulate the choice between RP and RS-exon SDs. First, analysis of Drosophila RP SD mutants demonstrates that SD competition suppresses inclusion of cryptic exons in endogenous contexts. Second, characterization of RS-exon reporters implicates exonic sequences as influencing choice of RS-exon usage. Using RS-exon swap and mutagenesis assays, we show exonic sequences can determine RS-exon inclusion. Finally, we provide evidence that splicing can suppress utilization of RP SDs to enable RS-exon expression. Overall, multiple factors can influence splicing of Drosophila RS-exons, which usually result in their complete suppression as zero-nucleotide RPs, but occasionally yield translated RS-exons.

Large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. However, Kavaler et al. show here that developmental specification toward a non-neuronal fate in the Drosophila melanogaster peripheral sensory organ lineage depends critically on mir-279/996 repression of the Notch repressor Insensible., Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. Here, we identify a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. We observe that binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath–neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, we identify Insensible (Insb), another neural nuclear Notch pathway inhibitor, as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.

Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of “conserved, essential” and “young, RNAi-lethal” genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.

Neural development is controlled at multiple levels to orchestrate appropriate choices of cell fate and differentiation. Although more attention has been paid to the roles of neural-restricted factors, broadly expressed factors can have compelling impacts on tissue-specific development. Here, we describe in vivo conditional knockout analyses of murine Ars2, which has mostly been studied as a general RNA-processing factor in yeast and cultured cells. Ars2 protein expression is regulated during neural lineage progression, and is required for embryonic neural stem cell (NSC) proliferation. In addition, Ars2 null NSCs can still transition into post-mitotic neurons, but fail to undergo terminal differentiation. Similarly, adult-specific deletion of Ars2 compromises hippocampal neurogenesis and results in specific behavioral defects. To broaden evidence for Ars2 as a chromatin regulator in neural development, we generated Ars2 ChIP-seq data. Notably, Ars2 preferentially occupies DNA enhancers in NSCs, where it colocalizes broadly with NSC regulator SOX2. Ars2 association with chromatin is markedly reduced following NSC differentiation. Altogether, Ars2 is an essential neural regulator that interacts dynamically with DNA and controls neural lineage development.

microRNAs (miRNAs) are a broad class of ~22 nucleotide regulatory RNA, which collectively have broad effects on the transcriptome and are involved in diverse biology, from development and adult physiology, and from homeostasis to disease and pathology. We investigated the effects of systematically expressing microRNAs (miRNAs) during the development of the Drosophila compound eye using the GMR-Gal4 driver. The objective was to determine what fraction of miRNAs were capable of inducing aberrant morphology that was easily and reproducibly scored by visual inspection under a dissecting microscope. We assayed multiple independent insertions of 166 miRNA transgenes (536 lines), comprising solo miRNAs, miRNA operons and individual constituent miRNAs from operons. We find a substantial number reproducibly altered normal eye development and a smaller number induced lethality in most or all progeny. We provide the comprehensive results of this screen, documenting numerous miRNA transgenes that interfered with normal eye development when activated using GMR-Gal4. These data can be mined by the Drosophila community to query the in vivo effects of any individual miRNA of interest in the eye, as well as utilized as a foundation for more complex genetic perturbations that involve miRNA misexpression in the eye.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22219-8

The tissue-specific deployment of highly extended neural 3’ UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well-understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9 and Fne) have similar capacities to induce a lengthened 3’ UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3’ UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne towards the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons, and may explain preferential evolutionary emergence of miRNA operons.

Summary Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

N[superscript 6]-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease., Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology (SMART)), National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES022858), National Institute of Environmental Health Sciences ( ES002109), National Science Foundation (U.S.) (MCB-1412379), German Science Foundation (Fellowship)

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that normal Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently de-represses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that substantially requires miRNA control.

Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-β in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.

Background Drosophila melanogaster has one of best-described transcriptomes of any multicellular organism. Nevertheless, the paucity of 3′-sequencing data in this species precludes comprehensive assessment of alternative polyadenylation (APA), which is subject to broad tissue-specific control. Results Here, we generate deep 3′-sequencing data from 23 developmental stages, tissues, and cell lines of D. melanogaster, yielding a comprehensive atlas of ~ 62,000 polyadenylated ends. These data broadly extend the annotated transcriptome, identify ~ 40,000 novel 3′ termini, and reveal that two-thirds of Drosophila genes are subject to APA. Furthermore, we dramatically expand the numbers of genes known to be subject to tissue-specific APA, such as 3′ untranslated region (UTR) lengthening in head and 3′ UTR shortening in testis, and characterize new tissue and developmental 3′ UTR patterns. Our thorough 3′ UTR annotations permit reassessment of post-transcriptional regulatory networks, via conserved miRNA and RNA binding protein sites. To evaluate the evolutionary conservation and divergence of APA patterns, we generate developmental and tissue-specific 3′-seq libraries from Drosophila yakuba and Drosophila virilis. We document broadly analogous tissue-specific APA trends in these species, but also observe significant alterations in 3′ end usage across orthologs. We exploit the population of functionally evolving poly(A) sites to gain clear evidence that evolutionary divergence in core polyadenylation signal (PAS) and downstream sequence element (DSE) motifs drive broad alterations in 3′ UTR isoform expression across the Drosophila phylogeny. Conclusions These data provide a critical resource for the Drosophila community and offer many insights into the complex control of alternative tissue-specific 3′ UTR formation and its consequences for post-transcriptional regulatory networks. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1358-0) contains supplementary material, which is available to authorized users.

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A ‘writer’ complex, validate its YTH ‘readers’ CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway., N 6-methyladenosine (m6A) is a conserved RNA modification that has recently emerged as an important regulator of messenger RNA processing and activity. Here, the authors provide evidence that m6A pathway facilitates female-specific splicing of Sxl, regulating sex determination in Drosophila.

Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.

To assess miRNA evolution across the Drosophila genus, we analyzed several billion small RNA reads across 12 fruit fly species. These data permit comprehensive curation of species- and clade-specific variation in miRNA identity, abundance, and processing. Among well-conserved miRNAs, we observed unexpected cases of clade-specific variation in 5′ end precision, occasional antisense loci, and putatively noncanonical loci. We also used strict criteria to identify a large set (649) of novel, evolutionarily restricted miRNAs. Within the bulk collection of species-restricted miRNAs, two notable subpopulations are splicing-derived mirtrons and testes-restricted, recently evolved, clustered (TRC) canonical miRNAs. We quantified miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover. We observed striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identified flux heterogeneity among Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. Mirtrons are associated with high rates of 3′ untemplated addition, a mechanism that impedes their biogenesis, whereas TRC miRNAs appear to evolve under positive selection. Altogether, these data reveal miRNA diversity among Drosophila species and principles underlying their emergence and evolution.

Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3′ exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce Drosophila as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure–function analysis of the Drosophila TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of Tailor and Dis3L2, which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis.

Major applications of RNA-seq data include studies of how the transcriptome is modulated at the levels of gene expression and RNA processing, and how these events are related to cellular identity, environmental condition, and/or disease status. While many excellent tools have been developed to analyze RNA-seq data, these generally have limited efficacy for annotating 3′ UTRs. Existing assembly strategies often fragment long 3′ UTRs, and importantly, none of the algorithms in popular use can apportion data into tandem 3′ UTR isoforms, which are frequently generated by alternative cleavage and polyadenylation (APA). Consequently, it is often not possible to identify patterns of differential APA using existing assembly tools. To address these limitations, we present a new method for transcript assembly, Isoform Structural Change Model (IsoSCM) that incorporates change-point analysis to improve the 3′ UTR annotation process. Through evaluation on simulated and genuine data sets, we demonstrate that IsoSCM annotates 3′ termini with higher sensitivity and specificity than can be achieved with existing methods. We highlight the utility of IsoSCM by demonstrating its ability to recover known patterns of tissue-regulated APA. IsoSCM will facilitate future efforts for 3′ UTR annotation and genome-wide studies of the breadth, regulation, and roles of APA leveraging RNA-seq data. The IsoSCM software and source code are available from our website https://github.com/shenkers/isoscm.

SummaryThe Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features [1, 2]. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals [3–10]. Here, we define the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion.

Many animal miRNA loci reside in genomic clusters that generate multicistronic primary-miRNA transcripts. While clusters that contain copies of the same miRNA hairpin are clearly products of local duplications, the evolutionary provenance of clusters with disparate members is less clear. Recently, it was proposed that essentially all such clusters in Drosophila derived from de novo formation of miRNA-like hairpins within existing miRNA transcripts, and that the maintenance of multiple miRNAs in such clusters was due to evolutionary hitchhiking on a major cluster member. However, this model seems at odds with the fact that many such miRNA clusters are composed of well-conserved miRNAs. In an effort to trace the birth and expansion of miRNA clusters that are presently well-conserved across Drosophilids, we analyzed a broad swath of metazoan species, with particular emphasis on arthropod evolution. Beyond duplication and de novo birth, we highlight a diversity of modes that contribute to miRNA evolution, including neofunctionalization of miRNA copies, fissioning of locally duplicated miRNA clusters, miRNA deletion, and miRNA cluster expansion via the acquisition and/or neofunctionalization of miRNA copies from elsewhere in the genome. In particular, we suggest that miRNA clustering by acquisition represents an expedient strategy to bring cohorts of target genes under coordinate control by miRNAs that had already been individually selected for regulatory impact on the transcriptome.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Alternative cleavage and polyadenylation (APA) can diversify coding and non-coding regions, but has particular impact on increasing 3' UTR diversity. Through the gain or loss of regulatory elements such as RNA binding protein and microRNA sites, APA can influence transcript stability, localization, and translational efficiency. Strikingly, the central nervous systems of invertebrate and vertebrate species express a broad range of transcript isoforms bearing extended 3' UTRs. The molecular mechanism that permits proximal 3' end bypass in neurons is mysterious, and only beginning to be elucidated. This landscape of neural 3' UTR extensions, many reaching unprecedented lengths, may help service the unique post-transcriptional regulatory needs of neurons. A combination of approaches, including transcriptome-wide profiling, genetic screening to identify APA factors, biochemical dissection of alternative 3' end formation, and manipulation of individual neural APA targets, will be necessary to gain fuller perspectives on the mechanism and biology of neural-specific 3' UTR lengthening.

Summary The Drosophila Bithorax complex (BX-C) Hox cluster contains a bidirectionally transcribed miRNA locus, and a deletion mutant (Δ 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 CNS. Δ 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, because sterility of Δ mir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in Δ mir females, and substantially rescued by heterozygosity of Δ 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.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features1, 2, 3, 4, 5, 6. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a ‘universal model’ based on a single set of organism-independent parameters. In particular, this work was funded by a contract from the National Human Genome Research Institute modENCODE Project, contract U01 HG004271 and U54 HG006944, to S.E.C. (principal investigator) and P.C., T.R.G., R.A.H. and B.R.G. (co-principal investigators) with additional support from R01 GM076655 (S.E.C.) both under Department of Energy contract no. DE-AC02-05CH11231, and U54 HG007005 to B.R.G. J.B.B.’s work was supported by NHGRI K99 HG006698 and DOE DE-AC02-05CH11231. Work in P.J.B.’s group was supported by the modENCODE DAC sub award 5710003102, 1U01HG007031-01 and the ENCODE DAC 5U01HG004695-04. Work in M.B.G.’s group was supported by NIH grants HG007000 and HG007355. Work in Bloomington was supported in part by the Indiana METACyt Initiative of Indiana University, funded by an award from the Lilly Endowment, Inc. Work in E.C.L.’s group was supported by U01-HG004261 and RC2-HG005639. P.J.P. acknowledges support from the National Institutes of Health (grant no. U01HG004258). We thank the HAVANA team for providing annotation of the human reference genome, whose work is supported by National Institutes of Health (grant no. 5U54HG004555), the Wellcome Trust (grant no. WT098051). R.G. acknowledges support from the Spanish Ministry of Education (grant BIO2011-26205). We also acknowledge use of the Yale University Biomedical High Performance Computing Center. R.W.'s lab was supported by grant no. U01 HG 004263.

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.

Skin stem cells (SCs) are specified and rapidly expanded to fuel body growth during early development. However, the molecular mechanisms that govern the amplification of skin SCs remain unclear. Here we report an essential role for miR-205, one of the most highly expressed microRNAs in skin SCs, in promoting neonatal expansion of these cells. Unlike most mammalian miRNAs, genetic deletion of miR-205 causes neonatal lethality with severely compromised epidermal and hair follicle growth. In the miR-205 knockout skin SCs, phospho-Akt is significantly downregulated, and the SCs prematurely exit the cell cycle. In the hair follicle, this accelerates the transition of the neonatal SCs towards quiescence. We identify multiple miR-205-targeted negative regulators of PI(3)K signalling that mediate the repression of phospho-Akt and restrict the proliferation of SCs. Our findings reveal an essential role for miR-205 in maintaining the expansion of skin SCs by antagonizing negative regulators of PI(3)K signalling.

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class.

MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila β-catenin) expression and activity by directly targeting the 3′-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.

microRNAs (miRNAs) are ~22 nucleotide (nt) RNAs that coordinate vast regulatory networks in animals, and thereby influence myriad processes. This review examines evidence that miRNAs play continuous roles in adults, in ways that are separable from developmental control. Adult-specific activities for miRNAs have been described in a variety of stem cell populations, in the context of neural function and cardiovascular biology, in metabolism and physiology, and during cancer. In addition to reviewing recent results, we also discuss methods for studying miRNA activities specifically in adults and evaluate their relative strengths and weaknesses. A fuller understanding of continuous functions of miRNAs in adults has bearing on efforts and opportunities to manipulate miRNAs for therapeutic purposes.

The activity of the Notch pathway revolves around a CSL-class transcription factor, which recruits distinct complexes that activate or repress target gene expression. The co-activator complex is deeply conserved and includes the cleaved Notch intracellular domain (NICD) and Mastermind. By contrast, numerous CSL co-repressor proteins have been identified, and these are mostly different between invertebrate and vertebrate systems. In this study, we demonstrate that mammalian BEND6 is a neural BEN-solo factor that shares many functional attributes with Drosophila Insensitive, a co-repressor for the Drosophila CSL factor. BEND6 binds the mammalian CSL protein CBF1 and antagonizes Notch-dependent target activation. In addition, its association with Notch- and CBF1-regulated enhancers is promoted by CBF1 and antagonized by activated Notch. In utero electroporation experiments showed that ectopic BEND6 inhibited Notch-mediated self-renewal of neocortical neural stem cells and promoted neurogenesis. Conversely, knockdown of BEND6 increased NSC self-renewal in wild-type neocortex, and exhibited genetic interactions with gain and loss of Notch pathway activity. We recapitulated all of these findings in cultured neurospheres, in which overexpression and depletion of BEND6 caused reciprocal effects on neural stem cell renewal and neurogenesis. These data reveal a novel mammalian CSL co-repressor in the nervous system, and show that the Notch-inhibitory activity of certain BEN-solo proteins is conserved between flies and mammals.

We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a “BEN-solo” protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN–DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family.

The deposited article is a post-print version and has peer review. This deposit is composed by the main article, and it hasn't any supplementary materials associated. The supplementary materials are present in the publisher's page in the following link: http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0960982213001899/1-s2.0-S0960982213001899-mmc1.pdf/272099/html/S0960982213001899/187ecb1b8f94027ceebb87907c58eb3f/mmc1.pdf Identifying the genetic mechanisms underlying phenotypic change is essential to understanding how gene regulatory networks and ultimately the genotype-to-phenotype map evolve. It is recognized that microRNAs (miRNAs) have the potential to facilitate evolutionary change [1-3]; however, there are no known examples of natural morphological variation caused by evolutionary changes in miRNA expression. Therefore, the contribution of miRNAs to evolutionary change remains unknown [1, 4]. Drosophila melanogaster subgroup species display a portion of trichome-free cuticle on the femur of the second leg called the "naked valley." It was previously shown that Ultrabithorax (Ubx) is involved in naked valley variation between D. melanogaster and D. simulans [5, 6]. However, naked valley size also varies among populations of D. melanogaster, ranging from 1,000 up to 30,000 μm(2). We investigated the genetic basis of intraspecific differences in the naked valley in D. melanogaster and found that neither Ubx nor shavenbaby (svb) [7, 8] contributes to this morphological difference. Instead, we show that changes in mir-92a expression underlie the evolution of naked valley size in D. melanogaster through repression of shavenoid (sha) [9]. Therefore, our results reveal a novel mechanism for morphological evolution and suggest that modulation of the expression of miRNAs potentially plays a prominent role in generating organismal diversity. Austrian Science Fund (FWF) grant: (M1059-B09); Oxford Brookes University; National Institute of General Medical Sciences of the National Institutes of Health grant: (R01-GM083300). info:eu-repo/semantics/publishedVersion

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions, we identified embryonic Grh targets by ChIP-seq and gene expression analysis. We show that Grh controls hundreds of target genes. Repression or activation correlates with the distance of Grh binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU-domain TF, Vvl, (ventral veins lacking) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. We conclude that Grh and Vvl participate in a regulatory network controlling epithelial maturation.

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs (“mirtrons”), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 5′-tailed mirtrons, and 3′-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.