Events In Drosophila Research Articles

Counting pseudoalignments to novel splicing events.

Alternative splicing (AS) of introns from pre-mRNA produces diverse sets of transcripts across cell types and tissues, but is also dysregulated in many diseases. Alignment-free computational methods have greatly accelerated the quantification of mRNA transcripts from short RNA-seq reads, but they inherently rely on a catalog of known transcripts and might miss novel, disease-specific splicing events. By contrast, alignment of reads to the genome can effectively identify novel exonic segments and introns. Event-based methods then count how many reads align to predefined features. However, an alignment is more expensive to compute and constitutes a bottleneck in many AS analysis methods. Here, we propose fortuna, a method that guesses novel combinations of annotated splice sites to create transcript fragments. It then pseudoaligns reads to fragments using kallisto and efficiently derives counts of the most elementary splicing units from kallisto's equivalence classes. These counts can be directly used for AS analysis or summarized to larger units as used by other widely applied methods. In experiments on synthetic and real data, fortuna was around 7× faster than traditional align and count approaches, and was able to analyze almost 300 million reads in just 15 min when using four threads. It mapped reads containing mismatches more accurately across novel junctions and found more reads supporting aberrant splicing events in patients with autism spectrum disorder than existing methods. We further used fortuna to identify novel, tissue-specific splicing events in Drosophila. fortuna source code is available at https://github.com/canzarlab/fortuna.

The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms.

The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, we identify an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain. We also demonstrate that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. Our findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.

Use of circular RNAs as markers of readthrough transcription to identify factors regulating cleavage/polyadenylation events.

Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.

Out of the testis, into the ovary: biased outcomes of gene duplication and deletion in Drosophila.

Gene turnover is a key source of adaptive variation. Yet most evolutionary studies have focused on gene duplication, dismissing gene deletion as a mechanism that simply eradicates redundancy. Here, I use genome-scale sequence and multi-tissue expression data from Drosophila melanogaster and Drosophila pseudoobscura to simultaneously assess the evolutionary outcomes of gene duplication and deletion in Drosophila. I find that gene duplication is more frequent than gene deletion in both species, indicating that it may play a more important role in Drosophila evolution. However, examination of several genic properties reveals that genes likely possess distinct functions after duplication that diverge further before deletion, suggesting that loss of redundancy cannot explain a majority of gene deletion events in Drosophila. Moreover, in addition to providing support for the well-known "out of the testis" origin of young duplicate genes, analyses of gene expression profiles uncover a preferential bias against deletion of old ovary-expressed genes. Therefore, I propose a novel "into the ovary" hypothesis for gene deletion in Drosophila, in which gene deletion may promote adaptation by salvaging genes that contribute to the evolution of female reproductive phenotypes. Under this combined "out of the testis, into the ovary" evolutionary model, gene duplication and deletion work in concert to generate and maintain a balanced repertoire of genes that promote sex-specific adaptation in Drosophila.

OvoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila.

Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1. In this system (“ovoD co-selection”), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.

Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy.

Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster. One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we apply the successful co-CRISPR technique in Drosophila to simultaneously target a gene of interest and a marker gene, ebony, which is a recessive gene that produces dark body color and has the further advantage of not being a commonly used transgenic marker. We found that Drosophila broods containing higher numbers of CRISPR-induced ebony mutations (“jackpot” lines) are significantly enriched for indel events in a separate gene of interest, while broods with few or no ebony offspring showed few mutations in the gene of interest. Using two different PAM sites in our gene of interest, we report that ∼61% (52–70%) of flies from the ebony-enriched broods had an indel in DNA near either PAM site. Furthermore, this marker mutation system may be useful in detecting the less frequent homology-directed repair events, all of which occurred in the ebony-enriched broods. By focusing on the broods with a significant number of ebony flies, successful identification of CRISPR-induced events is much faster and more efficient. The co-CRISPR technique we present significantly improves the screening efficiency in identification of genome-editing events in Drosophila.

Adhesion molecule Kirrel3/Neph2 is required for the elongated shape of myocytes during skeletal muscle differentiation.

Kirrel/Neph proteins are evolutionarily conserved members of the immunoglobulin superfamily of adhesion proteins. Kirrel3 is the mouse orthologue of Dumbfounded (Duf), a family member that regulates myoblast pre-fusion events in Drosophila. Yet, the role of Kirrel3 in mammalian myogenesis has not been demonstrated. Experiments performed here indicate that the mouse Kirrel3 protein regulates morphological changes of myoblasts that are required for their subsequent fusion into multinucleated myotubes. We show that Kirrel3 is transiently expressed at the tips of myocytes during early myoblast differentiation and that its expression is dependent on the myogenic transcription factor, MyoD. Kirrel3 is transported in vesicles into the plasma membrane and its extracellular domain is cleaved in a proteasome-dependent manner. C-terminal deletion mutant lacking most of the intracellular domain accumulates at cellular extensions, does not undergo extracellular cleavage and induces the formation of large cell aggregates. This result suggests that the processing of the extracellular domain is regulated by the receptor's intracellular region. Knock-down of Kirrel3 in primary muscle progenitor cells (MPCs) prevented spindle shape myocyte formation, and significantly reduced their fusion with other myocytes to form multinucleated myotubes. In addition, migration of Kirrel3-deficient MPCs was randomized relative to the directed migration of control MPCs. We conclude that mouse Kirrel3 is a myobast adhesion molecule which promotes the morphological change of rounded MPC to a spindle shaped myocyte that migrates in a directed fashion and participates in the tight interactions between myocytes prior to their fusion.

RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens.

When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens.

Interaction of light regimes and circadian clocks modulate timing of pre-adult developmental events in Drosophila

BackgroundCircadian clocks have been postulated to regulate development time in several species of insects including fruit flies Drosophila melanogaster. Previously we have reported that selection for faster pre-adult development reduces development time (by ~19 h or ~11%) and clock period (by ~0.5 h), suggesting a role of circadian clocks in the regulation of development time in D. melanogaster. We reasoned that these faster developing flies could serve as a model to study stage-specific interaction of circadian clocks and developmental events with the environmental light/dark (LD) conditions. We assayed the duration of three pre-adult stages in the faster developing (FD) and control (BD) populations under a variety of light regimes that are known to modulate circadian clocks and pre-adult development time of Drosophila to examine the role of circadian clocks in the timing of pre-adult developmental stages.ResultsWe find that the duration of pre-adult stages was shorter under constant light (LL) and short period light (L)/dark (D) cycles (L:D = 10:10 h; T20) compared to the standard 24 h day (L:D = 12:12 h; T24), long LD cycles (L:D = 14:14 h; T28) and constant darkness (DD). The difference in the duration of pre-adult stages between the FD and BD populations was significantly smaller under the three LD cycles and LL compared to DD, possibly due to the fact that clocks of both FD and BD flies are driven at the same pace in the three LD regimes owing to circadian entrainment, or are rendered dysfunctional under LL.ConclusionsThese results suggest that interaction between light regimes and circadian clocks regulate the duration of pre-adult developmental stages in fruit flies D. melanogaster.

Impact of age-associated increase in 2′-O-methylation of miRNAs on aging and neurodegeneration in Drosophila

MicroRNAs (miRNAs) are 20- to ∼24-nucleotide (nt) small RNAs that impact a variety of biological processes, from development to age-associated events. To study the role of miRNAs in aging, studies have profiled the levels of miRNAs with time. However, evidence suggests that miRNAs show heterogeneity in length and sequence in different biological contexts. Here, by examining the expression pattern of miRNAs by Northern blot analysis, we found that Drosophila miRNAs show distinct isoform pattern changes with age. Surprisingly, an increase of some miRNAs reflects increased 2'-O-methylation of select isoforms. Small RNA deep sequencing revealed a global increase of miRNAs loaded into Ago2, but not into Ago1, with age. Our data suggest increased loading of miRNAs into Ago2, but not Ago1, with age, indicating a mechanism for differential loading of miRNAs with age between Ago1 and Ago2. Mutations in Hen1 and Ago2, which lack 2'-O-methylation of miRNAs, result in accelerated neurodegeneration and shorter life span, suggesting a potential impact of the age-associated increase of 2'-O-methylation of small RNAs on age-associated processes. Our study highlights that miRNA 2'-O-methylation at the 3' end is modulated by differential partitioning of miRNAs between Ago1 and Ago2 with age and that this process, along with other functions of Ago2, might impact age-associated events in Drosophila.

Functional implications of Drosophila insulin-like peptides in metabolism, aging, and dietary restriction

The neuroendocrine architecture and insulin/insulin-like signaling (IIS) events in Drosophila are remarkably conserved. As IIS pathway governs growth and development, metabolism, reproduction, stress response, and longevity; temporal, spatial, and nutrient regulation of dilps encoding Drosophila insulin-like peptides (DILPs) provides potential mechanisms in modulating IIS. Of eight DILPs (DILP1–8) identified, recent studies have furthered our understanding of physiological roles of DILP2, DILP3, DILP5, and DILP6 in metabolism, aging, and responses to dietary restriction (DR), which will be the focus of this review. While the DILP producing IPCs of the brain secrete DILP2, 3, and 5, fat body produces DILP6. Identification of factors that influence dilp expression and DILP secretion has provided insight into the intricate regulatory mechanisms underlying transcriptional regulation of those genes and the activity of each peptide. Studies involving loss-of-function dilp mutations have defined the roles of DILP2 and DILP6 in carbohydrate and lipid metabolism, respectively. While DILP3 has been implicated to modulate lipid metabolism, a metabolic role for DILP5 is yet to be determined. Loss of dilp2 or adult fat body specific expression of dilp6 has been shown to extend lifespan, establishing their roles in longevity regulation. The exact role of DILP3 in aging awaits further clarification. While DILP5 has been shown associated with DR-mediated lifespan extension, contradictory evidence that precludes a direct involvement of DILP5 in DR exists. This review highlights recent findings on the importance of conserved DILPs in metabolic homeostasis, DR, and aging, providing strong evidence for the use of DILPs in modeling metabolic disorders such as diabetes and hyperinsulinemia in the fly that could further our understanding of the underlying processes and identify therapeutic strategies to treat them.

The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by beta-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galbeta1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galbeta1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcbeta1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcbeta1-3(GlcAbeta1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.

Identifying tissue specific alternative splicing events in Drosophila

Identifying tissue specific alternative splicing events in Drosophila

Sex determination in the silkworm, Bombyx mori: A female determinant on the W chromosome and the sex-determining gene cascade

In insects, the sex is determined completely by genetic mechanisms, which at least in somatic tissues, are cell autonomous. The sex of the silkworm, Bombyx mori, is strongly controlled by the presence of the W chromosome. Genetic studies using translocations and deletions of W suggested that a presumptive feminizing gene (Fem) is located in a limited region of the W chromosome. Recent genomic studies revealed a small number of potential candidates for the Fem gene in this region. In addition, a Bombyx homologue of the Drosophila sex determining gene doublesex has been identified on an autosome and analyzed. Whereas the Drosophila doublesex gene is regulated by activation of splicing in females, the Bombyx doublesex gene (Bmdsx) encodes female- and male-specific mRNAs regulated via male-specific repression of splicing. The vitellogenin gene (Vg) is a target of the BmDSX protein, which directly binds to the Vg promoter. Furthermore, as ectopic expression of the male-type Bmdsx induces male-like transformation of the sexual organs, BmDSX may control sex-specific morphological characteristics in Bombyx. This suggests that although upstream events in Drosophila and Bombyx sex determination differ, similarities between the two species do exist in downstream genetic control of sex determination.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

Splicing regulators: targets and drugs

Recent work has revealed a complex network of distinct alternative splicing events in Drosophila as well as drugs that interfere specifically and directly with one class of splicing regulators in human cells.

Epsin potentiatesNotchpathway activity inDrosophilaandC. elegans

Endocytosis and trafficking within the endocytosis pathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific proteins for regulated endocytosis. Here, we present a functional link between the Notch pathway and epsins. We identify the Drosophila ortholog of epsin, liquid facets (lqf), as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. We find that lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. We extended the link between the Notch pathway and epsin function to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. Our results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis.

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction.

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc(14) mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.

Arrow encodes an LDL-receptor-related protein essential for Wingless signalling.

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.

Evidence for functional differentiation among Drosophila septins in cytokinesis and cellularization.

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut(+) transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both.