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To survive, animals must convert sensory information into appropriate behaviours1,2. Vision is a common sense for locating ethologically relevant stimuli and guiding motor responses3–5. How circuitry converts object location in retinal coordinates to movement direction in body coordinates remains largely unknown. Here we show through behaviour, physiology, anatomy and connectomics in Drosophila that visuomotor transformation occurs by conversion of topographic maps formed by the dendrites of feature-detecting visual projection neurons (VPNs)6,7 into synaptic weight gradients of VPN outputs onto central brain neurons. We demonstrate how this gradient motif transforms the anteroposterior location of a visual looming stimulus into the fly’s directional escape. Specifically, we discover that two neurons postsynaptic to a looming-responsive VPN type promote opposite takeoff directions. Opposite synaptic weight gradients onto these neurons from looming VPNs in different visual field regions convert localized looming threats into correctly oriented escapes. For a second looming-responsive VPN type, we demonstrate graded responses along the dorsoventral axis. We show that this synaptic gradient motif generalizes across all 20 primary VPN cell types and most often arises without VPN axon topography. Synaptic gradients may thus be a general mechanism for conveying spatial features of sensory information into directed motor outputs.

Summary Precise patterns of synaptic connections between neurons are encoded in their genetic programs. Here, we use single-cell RNA sequencing to profile neuronal transcriptomes at multiple stages in the developing Drosophila visual system. We devise an efficient strategy for profiling neurons at multiple time points in a single pool, thereby minimizing batch effects and maximizing the reliability of time-course data. A transcriptional atlas spanning multiple stages is generated, including more than 150 distinct neuronal populations; of these, 88 are followed through synaptogenesis. This analysis reveals a common (pan-neuronal) program unfolding in highly coordinated fashion in all neurons, including genes encoding proteins comprising the core synaptic machinery and membrane excitability. This program is overlaid by cell-type-specific programs with diverse cell recognition molecules expressed in different combinations and at different times. We propose that a pan-neuronal program endows neurons with the competence to form synapses and that cell-type-specific programs control synaptic specificity.

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.

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.

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.

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.

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.

Drosophila Elav is the founding member of the conserved family of Hu RNA binding proteins (RBPs), which collectively play critical and diverse roles in post-transcriptional regulation. In particular, Elav has for >20 years served as the canonical neuronal marker, owing to the availability of specific monoclonal antibodies. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. In particular, mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows that not only does it generate extended 3' UTR isoforms, its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR region includes multiple conserved, high-affinity sites for the miR-279/996 family. Notably, out of several miRNA mutants tested, we find that endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996. We also observe cross-repression of Elav by another RBP derepressed in non-neural miRNA pathway clones, namely Mei-P26. Finally, we demonstrate that ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. It is critical to restrict Elav outside of the nervous system as misexpression of Elav in non-neural territories has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell-type specific expression of a conserved neural RBP.

Drosophila Elav is the founding member of the conserved family of Hu RNA-binding proteins (RBPs), which play crucial and diverse roles in post-transcriptional regulation. Elav has long served as the canonical neuronal marker. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. Mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows not only that it generates extended 3′ UTR isoforms, but also that its universal 3′ UTR isoform is much longer than previously believed. This longer common 3′ UTR includes multiple conserved, high-affinity sites for the miR-279/996 family. Of several miRNA mutants tested, endogenous Elav and a transgenic elav 3′ UTR sensor are derepressed in mutant clones of mir-279/996. We also observe cross-repression of Elav by Mei-P26, another RBP derepressed in non-neural miRNA pathway clones. Ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. Repression of Elav in non-neural territories is crucial as misexpression here has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell type-specific expression of a conserved neural RBP.

Background In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. Results The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC). Conclusion pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm.