The response of drosophila to bacterial and fungal infections involves two signaling pathways, Toll and Imd, which both activate members of the transcription factor NF-kappaB family. Here we have studied the global transcriptional response of flies to infection with drosophila C virus. Viral infection induced a set of genes distinct from those regulated by the Toll or Imd pathways and triggered a signal transducer and activator of transcription (STAT) DNA-binding activity. Genetic experiments showed that the Jak kinase Hopscotch was involved in the control of the viral load in infected flies and was required but not sufficient for the induction of some virus-regulated genes. Our results indicate that in addition to Toll and Imd, a third, evolutionary conserved innate immunity pathway functions in drosophila and counters viral infection.

The fruit fly Drosophila melanogaster is a model system for studying innate immunity, including antiviral host defense. Infection with drosophila C virus triggers a transcriptional response that is dependent in part on the Jak kinase Hopscotch. Here we show that successful infection and killing of drosophila with the insect nodavirus flock house virus was strictly dependent on expression of the viral protein B2, a potent inhibitor of processing of double-stranded RNA mediated by the essential RNA interference factor Dicer. Conversely, flies with a loss-of-function mutation in the gene encoding Dicer-2 (Dcr-2) showed enhanced susceptibility to infection by flock house virus, drosophila C virus and Sindbis virus, members of three different families of RNA viruses. These data demonstrate the importance of RNA interference for controlling virus replication in vivo and establish Dcr-2 as a host susceptibility locus for virus infections.

Drosophila, like other invertebrates and plants, relies mainly on RNA interference for its defense against viruses. In flies, viral infection also triggers the expression of many genes. One of the genes induced, Vago, encodes a 18-kilodalton cysteine-rich polypeptide. Here we provide genetic evidence that the Vago gene product controlled viral load in the fat body after infection with drosophila C virus. Induction of Vago was dependent on the helicase Dicer-2. Dicer-2 belongs to the same DExD/H-box helicase family as do the RIG-I-like receptors, which sense viral infection and mediate interferon induction in mammals. We propose that this family represents an evolutionary conserved set of sensors that detect viral nucleic acids and direct antiviral responses.

Comparative genomics has revealed that major vertebrate lineages contain quantitatively and qualitatively different populations of retrotransposable elements and DNA transposons, with important differences also frequently observed between species of the same lineage. This is essentially due to (i) the differential evolution of ancestral families of transposable elements, with evolutionary scenarios ranging from complete extinction to massive invasion; (ii) the lineage-specific introduction of transposable elements by infection and horizontal transfer, as exemplified by endogenous retroviruses; and (iii) the lineage-specific emergence of new transposable elements, as particularly observed for non-coding retroelements called short interspersed elements (SINEs). During vertebrate evolution, transposable elements have repeatedly contributed regulatory and coding sequences to the host, leading to the emergence of new lineage-specific gene regulations and functions. In all vertebrate lineages, there is evidence of transposable element-mediated genomic rearrangements such as insertions, deletions, inversions and duplications potentially associated with or subsequent to speciation events. Taken together, these observations indicate that transposable elements are major drivers of genomic and biological diversity in vertebrates, with possible important roles in speciation and major evolutionary transitions.

Poeciliids, a family of live-bearing freshwater fish, including among others platyfish, swordtails and guppies, fully illustrate the diversity of genetic sex determination mechanisms observed in teleosts. Besides unisexuality, a variety of sex-determining systems has been described in this group of fish, including male and female heterogamety with or without autosomal influence, as well as more complicated situations such as multichromosomal and polyfactorial sex determination. Due to the presence of different mechanisms in closely related species or even between populations within a same species, poeciliids are a very attractive model to study the evolutionary dynamics of sex determination. For one species, the Southern platyfish Xiphophorus maculatus, positional cloning of the master sex-determining gene has been initiated through the construction and sequencing of bacterial artificial chromosome contigs covering the region differentiating the X from the Y chromosome. Initial analysis revealed a high plasticity of the sex-determining region and the absence of synteny with other fish and vertebrate sex chromosomes, indicating an independent evolutionary origin.

BackgroundMembers of the makorin (mkrn) gene family encode RING/C3H zinc finger proteins with U3 ubiquitin ligase activity. Although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution.ResultsVia partial sequencing of a testis cDNA library from the poeciliid fish Xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn4. In addition to the already described mkrn1 and mkrn2, mkrn4 is the third example of a makorin gene present in both tetrapods and ray-finned fish. However, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. Mkrn2 and mkrn4 are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. Intriguingly, many mkrn1 and mkrn2 intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. The nature and number of zinc fingers were found to be conserved in Mkrn1 and Mkrn2 but much more variable in Mkrn4, with lineage-specific differences. RT-qPCR analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish (ray-finned fishes) and amphibians, but a strong relaxation of this specificity in birds and mammals. All three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos.ConclusionOur analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. From the three major vertebrate mkrn genes, mkrn4 shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos.

Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid- and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering ϳ30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.Alternative splicing of pre-mRNA transcripts allows a gene to code for more than one protein (1, 2). Very often similar but distinct versions of the same protein are made from a gene in this way. When alternative splicing is controlled in a cell typespecific manner, cells benefit from the particular version of a protein best suited to their needs. The biological importance of this is underlined by numerous observations linking impaired control of alternative splicing to human diseases, including cancer (3). Understanding splicing control will thus shed light on important aspects of normal and pathological cell life.In mammalian systems, alternative splicing control is often very complex and is characterized by multiple combinatorial control (4). For example, splicing of the neuron-specific exon N1 of the mouse c-src gene is activated by a purine-rich exonic sequence and an intronic splicing enhancer; the latter contains binding sites for hnRNP 1 H, hnRNP F, and KH-type splicing regulatory protein (5). Polypyrimidine tract binding protein (PTB) binds to intron sites flanking the N1 exon to repress splicing, and one of these lies within the enhancer (6). A neurally enriched homologue of PTB binds more stably to this site than does PTB but is a weaker repressor of splicing in vitro. A neurally enriched homologue of PTB also greatly enhances the binding of hnRNP H and KH-type splicing-regulatory protein to the enhancer (5). The N1 exon is thus under control of both activators and repressors, and their relative influence determines its splicing efficiency (7).We and others have investigated another example of multiple combinatorial control: splicing of the FGFR-2 gene K-SAM alternative exon, an exon spliced by epithelial cells. Splicing requires the action of...