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Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs.

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.

Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole agingDrosophila. We characterize 163 distinct cell types and perform an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further develop aging clock models to predict the fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find unique cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.

Outcomes in autologous breast reconstruction continue to improve with refinements in microsurgical techniques; however, donor-site morbidity remains a concern. Closed-incision negative pressure therapy (ciNPT) has been shown to reduce wound complications. Limited evaluation in abdominal donor sites has shown promising results. We hypothesize that ciNPT will reduce abdominal donor-site complications.A retrospective chart review was performed of patients who underwent abdominally based autologous free tissue transfer for breast reconstruction by 4 microsurgeons at an academic institution from 2015 to 2020. The application of a commercial ciNPT for donor-site management was at the discretion of the operating surgeon. Demographics, operative details, and management of donor-site complications were analyzed.Four hundred thirty-three patients underwent autologous breast reconstruction; 212 abdominal donor sites were managed with ciNPT and 219 with standard dressings. Demographics were statistically similar between groups. Abdominal wound healing complications were noted in 30.2% of ciNPT patients (64/212) and 22.8% of control patients (50/219, P = 0.08); however, overall wound complications were attributed to obesity on multivariable analysis. Closed-incision negative pressure therapy significantly decreased complications requiring reoperation (ciNPT 6.2%, 4/64; control 26.5%, 13/51; P = 0.004). There were no significant differences in surgical site infection rates (P = 0.73) and rates of abdominal scar revisions (ciNPT 11.8%, 25/212; control 9.1%, 20/219; P = 0.37).Use of ciNPT in abdominal donor-site management significantly decreases the incidence of delayed wound healing requiring surgical intervention, with one major wound healing complication prevented for every 6 donor sites managed with ciNPT.

Effective suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. InD. melanogaster,flamencois a master suppressor of TEs, preventing their movement from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, that is metabolized into Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity.flamencois thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find thatflamencohas undergone radical transformations in sequence content and even copy number acrosssimulansclade Drosophilid species.D. simulans flamencohas duplicated and diverged, and neither copy exhibits synteny withD. melanogasterbeyond the core promoter. Moreover,flamencoorganization is highly variable acrossD. simulansindividuals. Next, we find thatD. simulansandD. mauritiana flamencodisplay signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual stranded clusters. Overall, the physical and functional diversity offlamencoorthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

Although the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. Here, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans bears compelling signatures of sex chromosome conflict and provides insight into molecular strategies that underlie hpRNA emergence. In particular, our data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, recurrent targeting of testis HMG Box loci by hpRNAs, and connections to the piRNA pathway. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since we observe strong derepression of targets of the youngest hpRNAs, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to the segregation of species.

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.

Background Radiation creates significant challenges for breast reconstruction. There is no consensus regarding optimal timing for autologous reconstruction following radiation. This study explores clearly defined, shorter time intervals between completion of radiation and reconstruction than previously reported. Methods A retrospective review was performed on patients who underwent autologous reconstruction by five microsurgeons at an academic institution from 2009 to 2020. Cohorts were selected by time elapsed between radiation and autologous reconstruction including 24 months. Analysis compared baseline characteristics, operative details, complications, revision rates, and BREAST-Q scores. Analysis of variance was used for continuous variables and chi-square for discrete variables. Results In total, 462 radiated patients underwent 717 flaps. There were 69 patients at 24 months (26.6%). Age, time from mastectomy, and failure of primary reconstruction were higher at >24 months (p Conclusion Reconstruction can be safely performed within 3 months after radiation without increases in intraoperative, acute, or late reconstructive complications.

Left lateral sectionectomy (LLS) is one of the most commonly performed minimally invasive liver resections. While laparoscopic (L)-LLS is a well-established technique, over traditional open resection, it remains controversial if robotic (R)-LLS provides any advantages of L-LLS.A post hoc analysis of 997 patients from 21 international centres undergoing L-LLS or R-LLS from 2006 to 2020 was conducted. A total of 886 cases (214 R-LLS, 672 L-LLS) met study criteria. 1:1 and 1:2 propensity score matched (PSM) comparison was performed between R-LLSL-LLS. Further subset analysis by Iwate difficulty was also performed. Outcomes measured include operating time, blood loss, open conversion, readmission rates, morbidity and mortality.Comparison between R-LLS and L-LLS after PSM 1:2 demonstrated statistically significantly lower open conversion rate in R-LLS than L-LLS (0.6% versus 5%, p = 0.009) and median blood loss was also statistically significantly lower in R-LLS at 50 (80) versus 100 (170) in L-LLS (p = 0.011) after PSM 1:1 although there was no difference in the blood transfusion rate. Pringle manoeuvre was also found to be used more frequently in R-LLS, with 53(24.8%) cases versus to 84(12.5%) L-LLS cases (p 0.001). There was no significant difference in the other key perioperative outcomes such as operating time, length of stay, postoperative morbidity, major morbidity and 90-day mortality between both groups.R-LLS was associated with similar key perioperative outcomes compared to L-LLS. It was also associated with significantly lower blood loss and open conversion rates compared to L-LLS.

Controversies exist among liver surgeons regarding clinical outcomes of the laparoscopic versus the robotic approach for major complex hepatectomies. The authors therefore designed a study to examine and compare the perioperative outcomes of laparoscopic left hepatectomy or extended left hepatectomy (L-LH/L-ELH) versus robotic left hepatectomy or extended left hepatectomy (R-LH/R-ELH) using a large international multicenter collaborative database.An international multicenter retrospective analysis of 580 patients undergoing L-LH/L-ELH or R-LH/R-ELH at 25 specialized hepatobiliary centers worldwide was undertaken. Propensity score-matching (PSM) was used at a 1:1 nearest-neighbor ratio according to 15 perioperative variables, including demographics, tumor characteristics, Child-Pugh score, presence of portal hypertension, multiple resections, histologic diagnosis, and Iwate difficulty grade.Before the PSM, 190 (32 %) patients underwent R-LH/R-ELH, and 390 (68 %) patients underwent L-LH/L-ELH. After the matching, 164 patients were identified in each arm without significant differences in demographics, preoperative variables, medical history, tumor pathology, tumor characteristics, or Iwate score. Regarding intra- and postoperative outcomes, the rebotic approach had significantly less estimated blood loss (EBL) (100 ml [IQR 200 ml] vs 200 ml [IQR 235 ml]; p = 0.029), fewer conversions to open operations (n = 4 [2.4 %] vs n = 13, [7.9 %]; p = 0.043), and a shorter hospital stay (6 days [IQR 3 days] vs 7 days [IQR 3.3 days]; p = 0.009).Both techniques are safe and feasible in major hepatic resections. Compared with L-LH/L-ELH, R-LH/R-ELH is associated with less EBL, fewer conversions to open operations, and a shorter hospital stay.

Background Perforators are typically found in rows in the deep inferior epigastric perforator (DIEP) flap. As methods to assess flap perfusion continue to improve, surgeons may be more likely to select perforators traditionally avoided. The purpose of this article is to describe clinical outcomes based on row and number of perforators to reevaluate flap and abdominal donor site morbidity. Methods A retrospective analysis was performed on patients who underwent breast reconstruction with DIEP flaps by four microsurgeons from 2013 to 2020. The row and number of perforators were determined from operative reports. Chi-square and t-test or nonparametric Fisher's exact test and Wilcoxon two-sample test were used for discrete and continuous variable, respectively, as applicable. Logistic regression was used for multivariable analyses. Results Of 628 flaps, 305 were medial row (58.7%), 159 were lateral row (30.6%), and 55 had both rows (10.6%). Partial flap loss was higher in both rows (p = 0.003). Fat necrosis was higher with medial (p = 0.03) and both rows (p = 0.01) when compared with lateral using multivariable analysis. Hernia or bulge was higher in lateral row flaps (lateral: 8/157, 5.1%; medial, 5/299, 1.7%; both, 0/55; p = 0.05); however, mesh was more commonly used in both row flaps (p = 0.05). There was no difference in fat necrosis or abdominal morbidity between single and multiple perforators. Conclusion There was no difference in fat necrosis based on the number or row of perforators. The lateral row provides adequate perfusion but may be associated with an elevated risk of hernia or bulge. Patients may benefit from mesh, especially when both rows are dissected.

The process of alternative polyadenylation (APA) generates multiple 3' UTR isoforms for a given locus, which can alter regulatory capacity and on occasion change coding potential. APA was initially characterized for a few genes, but in the past decade, has been found to be the rule for metazoan genes. While numerous differences in APA profiles have been catalogued across genetic conditions, perturbations, and diseases, our knowledge of APA mechanisms and biology is far from complete. In this review, we highlight recent findings regarding the role of the conserved ELAV/Hu family of RNA binding proteins (RBPs) in generating the broad landscape of lengthened 3' UTRs that is characteristic of neurons. We relate this to their established roles in alternative splicing, and summarize ongoing directions that will further elucidate the molecular strategies for neural APA, the in vivo functions of ELAV/Hu RBPs, and the phenotypic consequences of these regulatory paradigms in neurons.

Background: Despite the rapid advances that minimally invasive liver resection has gained in recent decades, open conversion is still inevitable in some circumstances. In this study, we aimed to determine the risk factors for open conversion after minimally invasive left lateral sectionectomy, and its impact on perioperative outcomes. Methods: This is a post hoc analysis of 2,445 of 2,678 patients who underwent minimally invasive left lateral sectionectomy at 45 international centers between 2004 and 2020. Factors related to open conversion were analyzed via univariate and multivariate analyses. One-to-one propensity score matching was used to analyze outcomes after open conversion versus non-converted cases. Results: The open conversion rate was 69/2,445 (2.8%). On multivariate analyses, male gender (3.6% vs 1.8%, P = .011), presence of clinically significant portal hypertension (6.1% vs 2.6%, P = .009), and larger tumor size (50 mm vs 32 mm, P < .001) were identified as independent factors associated with open conversion. The most common reason for conversion was bleeding in 27/69 (39.1%) of cases. After propensity score matching (65 open conversion vs 65 completed via minimally invasive liver resection), the open conversion group was associated with increased operation time, blood transfusion rate, blood loss, and postoperative stay compared with cases completed via the minimally invasive approach. Conclusion: Male sex, portal hypertension, and larger tumor size were predictive factors of open conversion after minimally invasive left lateral sectionectomy. Open conversion was associated with inferior perioperative outcomes compared with non-converted cases.

Most genes in higher eukaryotes express isoforms with distinct 3’ untranslated regions (3’ UTRs), generated by alternative polyadenylation (APA). Since 3’ UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3’-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3’ UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3’ UTR lengthening in differentiated neurons and 3’ UTR shortening in spermatocytes and spermatids. We trace dynamic 3’ UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3’ isoforms across the major cell types of this widely-studied model organism.

Retrospective studies evaluating tissue oximetry in a more recent cohort have shown superiority in flap outcomes. This study compares the use of tissue oximetry in a historical cohort to clinical observation and handheld doppler in a more recent cohort. We hypothesize that there is no benefit to using tissue oximetry.A retrospective review was performed on patients who underwent abdominal-based autologous breast reconstruction by five microsurgeons at an academic institution from 2009 to 2020. Method of postoperative flap monitoring was determined then operative details and complications were analyzed.1367 flaps were reviewed; 740 flaps in 460 patients were monitored with clinical observation and tissue oximetry, and 627 flaps in 391 patients were monitored with clinical observation and handheld doppler. There were no statistical differences in ischemic (p = .59) or congestive complications (p = .41), flap salvage rates when exploring for venous or arterial compromise (p = .52), or early flap loss (p = .56). Although not significant, acute flap-related return to the operating room was lower in the doppler group (4.6%) compared to the oximetry group (6.1%; p = .22). Flaps monitored with tissue oximetry had a statistical increase in length of stay (4.8 ± 1.4 days vs. 3.8 ± 1.6 days; p ≤ .001). The rates of late partial flap loss and fat necrosis were significantly higher in the oximetry group (2.6%, 19/740 vs. 0.3%, 2/740; p = .04) and (18.2%, 135/740 vs. 13.6%, 85/627; p = .02), respectively.There is no statistical benefit to the use of tissue oximetry compared to handheld doppler in flap monitoring with regards to flap outcomes.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known forDrosophilafactors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

miR-486 is a muscle-enriched microRNA, or “myomiR,” that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized mir-486 knockout mice (mir-486 KO). mir-486 KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in mir-486 KO:mdx5cv (DKO) mice. To identify direct in vivo miR-486 muscle target transcripts, we integrated RNA sequencing and chimeric miRNA eCLIP sequencing to identify key transcripts and pathways that contribute towards mir-486 KO and dystrophic disease pathologies. These targets included known and novel muscle metabolic and dystrophic structural remodeling factors of muscle and skeletal muscle contractile transcript targets. Together, our studies identify miR-486 as essential for normal muscle function, a driver of pathological remodeling in dystrophin-deficient muscle, a useful biomarker for dystrophic disease progression, and highlight the use of multiple omic platforms to identify in vivo microRNA target transcripts.

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.

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.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have been shown to play essential roles in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. MicroRNA-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. In comparison to our DMD mouse muscle transcriptomes, we identified several of these miR-486 muscle targets including known modulators of dystrophinopathy disease symptoms. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts.Abstract Figure

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.

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.

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.

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.

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.

The recent beyond-exponential growth in diverse collections of deep sequencing datasets creates enormous opportunities for discovery, concomitant with new challenges for displaying and interpreting these data. Notably, the availability of scores of whole genome sequences in multiple species clades enables comparative studies of functional elements. However, current genome browsers do not permit effective visualization of multigenome experimental data. Here, we present CrossBrowse, a standalone desktop application for displaying and browsing cross-species genomic datasets. We utilize data standards and graphic representation of popular browsers, and incorporate an intuitive graphical visualization of genome synteny that facilitates and drives human interrogation of comparative data. Our platform permits users with minimal informatics capacity to select arbitrary sets of genomes for display, upload and configure multiple datasets, and interact with vertebrate-sized genomic datasets in real-time. We illustrate the utility of CrossBrowse with interrogation of comparative invertebrate and mammalian datasets that provide insights into diverse aspects of transcriptional and post-transcriptional regulation. Of note, we show examplars of both preservation and divergence of functional elements that cannot be inferred from sequence alignments alone. Moreover, we demonstrate how inspection of primary data using CrossBrowse exposes an artifact in a typical strategy for assigning species-specific functional elements, and drives the implementation of an improved computational strategy. We anticipate that CrossBrowse will greatly foster user-based discovery within multispecies genomic datasets, and inform their bioinformatic interpretation.

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.

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that 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 derepresses 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 involves substantial miRNA control.

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.

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.