The IMD pathway induces the innate immune response to infection by gram-negative bacteria. We demonstrate strong female-to-male sex transformations in double mutants of the IMD pathway in combination with Doa alleles. Doa encodes a protein kinase playing a central role in somatic sex determination through its regulation of alternative splicing of dsx transcripts. Transcripts encoding two specific Doa isoforms are reduced in Rel null mutant females, supporting our genetic observations. A role for the IMD pathway in somatic sex determination is further supported by the induction of female-to-male sex transformations by Dredd mutations in sensitized genetic backgrounds. In contrast, mutations in either dorsal or Dif, the two other NF-κB paralogues of Drosophila, display no effects on sex determination, demonstrating the specificity of IMD signaling. Our results reveal a novel role for the innate immune IMD signaling pathway in the regulation of somatic sex determination in addition to its role in response to microbial infection, demonstrating its effects on alternative splicing through induction of a crucial protein kinase.

Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA-binding protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA-binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA, and TRA2 are thought to not function in males. To better understand sex-specific regulation of gene expression, we analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. Our data uncouple the effects of Sxl and tra/tra2 in females in the-sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. We show that FNE protein levels are downregulated by Sxl in female heads, also independently of tra/tra2. We argue that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function.

The advent of high-throughput technologies to analyze RNA expression levels and transcript structure has brought renewed attention to the age-old question of what differentiates males from females. In Drosophila, the characterized somatic sex determination cascade includes proteins implicated in the regulation of pre-mRNA splicing as well as at least 2 transcription factors at its base. Both DNA microarrays and RNA-Seq have been applied in a number of studies to determine the identities, expression levels and structure of transcripts expressed differentially in the 2 sexes, with remarkably divergent results in the number, structure and identity of affected transcripts. We briefly summarize these reports and discuss the reasons for the apparent discrepancies based upon the different conditions used for sample preparation and data analysis.

Background: The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment.Results: We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb umd.edu/software/spanki.Conclusions: Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

The fne (found‐in‐neurons) locus encodes one of the three paralogs of the ELAV gene family of Drosophila melanogaster. Members of this family are found throughout metazoans and encode RNA‐binding proteins with primarily neuronal localization, but with remarkably diverse functions given their high level of amino acid sequence conservation. The first identified member of the family, elav of Drosophila is a vital gene. Mutations in the second Drosophila elav paralog, rbp9, are viable but female sterile. No alleles of fne were previously available. FNE protein is normally present in the cytoplasm of all neurons throughout development. Here we describe the generation and characterization of fnenull mutations by homologous recombination. In contrast to elav and similar to rbp9, fnenull mutants are viable, but exhibit a specific and fully penetrant fusion of the β‐lobes in their mushroom bodies (MB), a paired neuropil of the central brain involved in a variety of complex behaviors. Mutant males have reduced courtship indices, but normal short‐ and long‐term courtship memory. Our data show that fne has specific functions which are non‐overlapping with the other two family members, namely in courtship behavior and in the development of the adult MB. The data further show that courtship memory does not require intact β‐lobes in the MB.

DOA kinase, the Drosophila member of the LAMMER/Clk protein kinase family, phosphorylates SR and SR-like proteins, including TRA, TRA2 and RBP1, which are responsible for the alternative splicing of transcripts encoding the key regulator of sex-specific expression in somatic cells of the fly, DOUBLESEX. Specific Doa alleles induce somatic female-to-male sex transformations, which can be enhanced when combined with mutations in loci encoding SR and SR-like proteins. The Doa locus encodes six different kinases, of which a 69-kDa isoform is expressed solely in females. Expression of this isoform is itself under the regulation of the somatic sex determination regulatory network, thus forming a putative positive autoregulatory loop which would reinforce the choice of the female cell-fate. We speculate that this loop is part of the evolutionary ancestral sex-determination machinery, based upon evidence demonstrating the existence of an autoregulatory loop involving TRA and TRA2 in several other insect species.

BACKGROUND:The Drosophila gene embryonic lethal abnormal visual system (elav) is the prototype of a gene family present in all metazoans. Its members encode structurally conserved neuronal proteins with three RNA Recognition Motifs (RRM) but they paradoxically act at diverse levels of post-transcriptional regulation. In an attempt to understand the history of this family, we searched for orthologs in eleven completely sequenced genomes, including those of humans, D. melanogaster and C. elegans, for which cDNAs are available.RESULTS:We analyzed 23 orthologs/paralogs of elav, and found evidence of gain/loss of gene copy number. For one set of genes, including elav itself, the coding sequences are free of introns and their products most resemble ELAV. The remaining genes show remarkable conservation of their exon organization, and their products most resemble FNE and RBP9, proteins encoded by the two elav paralogs of Drosophila. Remarkably, three of the conserved exon junctions are both close to structural elements, involved respectively in protein-RNA interactions and in the regulation of sub-cellular localization, and in the vicinity of diverse sequence variations.CONCLUSION:The data indicate that the essential elav gene of Drosophila is newly emerged, restricted to dipterans and of retrotransposed origin. We propose that the conserved exon junctions constitute potential sites for sequence/function modifications, and that RRM binding proteins, whose function relies upon plastic RNA-protein interactions, may have played an important role in brain evolution.

The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3'-untranslated region (3'-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3'-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K-d 40 nM). This finding supports our model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. We narrowed down the best binding site to a 20 nt long sequence A(U-5)A(U-3)G(U-2)A(U-6) in an alternative 3' exon. We propose and test a model in which the regulated use of this alternative 3' exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne.

elav, a gene necessary for neuronal differentiation and maintenance in Drosophila, encodes the prototype of a family of conserved proteins involved in post-transcriptional regulation. We identified found in neurons (fne), a gene encoding a new ELAV paralogue. We showed that FNE binds RNA in vitro. fne transcripts are present throughout development and contain long untranslated regions. Transcripts and proteins are restricted to neurons of the CNS and PNS during embryogenesis. These features are reminiscent of elav. However, fne expression is delayed compared to elav's, and FNE protein appears cytoplasmic, while ELAV is nuclear. GAL4-directed overexpression of fne in neurons leads to a reduction of stable transcripts produced from both the fne and elav endogenous loci, suggesting that fne autoregulates and also regulates elav.

genesisVolume 34, Issue 1-2 p. 71-73 UAS LinesFree Access Characterization of fly strains permitting GAL4-directed expression of found in neurons Fabienne Chalvet, Fabienne Chalvet Embryologie Moléculaire et Expérimentale, Université Paris Sud, Orsay, FranceSearch for more papers by this authorMarie-Laure Samson, Corresponding Author Marie-Laure Samson Mlaure.Samson@ibaic.u-psud.fr Embryologie Moléculaire et Expérimentale, Université Paris Sud, Orsay, FranceDéveloppement Signalisation et Cancer, UPRES-A 8080, Bâtiment 445, Université Paris Sud, 91405 Orsay Cedex, FranceSearch for more papers by this author Fabienne Chalvet, Fabienne Chalvet Embryologie Moléculaire et Expérimentale, Université Paris Sud, Orsay, FranceSearch for more papers by this authorMarie-Laure Samson, Corresponding Author Marie-Laure Samson Mlaure.Samson@ibaic.u-psud.fr Embryologie Moléculaire et Expérimentale, Université Paris Sud, Orsay, FranceDéveloppement Signalisation et Cancer, UPRES-A 8080, Bâtiment 445, Université Paris Sud, 91405 Orsay Cedex, FranceSearch for more papers by this author First published: 12 September 2002 https://doi.org/10.1002/gene.10108Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume34, Issue1-2Special Issue: GAL4/UAS in Drosophila September ‐ October 2002Pages 71-73 ReferencesRelatedInformation

The Drosophila locus embryonic lethal abnormal visual system (elav) encodes a nuclear RNA-binding protein essential for normal neuronal differentiation and maintenance of neurons. ELAV is thought to play its role by binding to RNAs produced by other genes necessary for neuronal differentiation and consequently to affect their metabolism by an as yet unknown mechanism. ELAV structural homologues have been identified in a wide range of organisms, including humans, indicating an important conserved role for the protein. Analysis of elav germline transformants presented here shows that one copy of elav minigenes lacking a complete 3' untranslated region (3' UTR) rescues null mutations at elav, but that two copies are lethal. Additional in vivo experiments demonstrate that elav expression is regulated through the 3' UTR of the gene and indicate that this level of regulation is dependent upon ELAV itself. Because ELAV is an RNA-binding protein, the simplest model to account for these findings is that ELAV binds to the 3' UTR of its own RNA to autoregulate its expression. I discuss the implications of these results for normal elav function.

We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5'-flanking region (54-55 bp), the 3'-flanking region (21-22 bp), and the internal spacer (149-206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6-17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps.

Regulated gene activity is crucial to the formation and function of the nervous system. It is well known that gene regulation can occur at the transcriptional, post-transcriptional, translational, and post-translational levels. In this review our focus has been on the post-transcriptional regulation in neurons and on neural-specific RNA binding proteins that may be involved in post-transcriptional modulation of gene activity. We have taken advantage of this opportunity to review our work on the elav gene of Drosophila melanogaster which encodes a neural-specific RNA binding protein and relate it to other members of this elav-like gene family. We report new data that suggests that elav is post-transcriptionally regulated and we demonstrate that below-threshold levels of ELAV protein severely affects neuronal differentiation.

The Ubx and abd-A gene products are required for proper development of thoracic and abdominal structures in Drosophila. We expressed LexA-Ubx and LexA-abdA fusion proteins in yeast. These proteins activated expression of target genes that carried either upstream LexA operators or upstream Ubx binding sites. Both proteins contain homeodomains. Experiments with mutant fusion proteins show that the homeodomain is not required for the proteins to form dimers or enter the nucleus, and that, when DNA binding is provided by the LexA moiety, the homeodomain is not required for gene activation. Our results suggest that the homeodomain is necessary for these proteins to bind Ubx sites, but that the homeodomain does not contact DNA exactly like bacterial helix-turn-helix proteins. Finally, our data suggest that gene activation by these proteins is a simple consequence of their binding to DNA, while negative gene regulation requires that these proteins act together with other Drosophila gene products.

The 5S genes of the eight species of the D. melanogaster subgroup have been mapped. The spacers, in contrast with coding regions, differ markedly between most species. One 5S gene unit has been sequenced for both D. simulans and D. teissieri. The mature 5S RNA region in these two species is identical to the corresponding region of D. melanogaster. Only 5 nucleotide variations occur between the D. melanogaster and D. simulans 5S gene spacers. The spacer in D. teissieri is very different. Only two segments, located one at each side of the coding region, are clearly homologous to corresponding sequences of D. melanogaster and D. simulans.