MORE THAN ONE WAY TO PRODUCE PROTEIN DIVERSITY: DUPLICATION AND LIMITED ALTERNATIVE SPLICING OF AN ADHESION MOLECULE GENE IN BASAL ARTHROPODS

Exon duplication and alternative splicing evolved multiple times in metazoa and are of overall importance in shaping genomes and allowing organisms to produce many fold more proteins than there are genes in the genome. No other example is as striking as the one of the Down syndrome cell adhesion molecule (Dscam) of insects and crustaceans (pancrustaceans) involved in the nervous system differentiation and in the immune system. To elucidate the evolutionary history of this extraordinary gene, we investigated Dscam homologs in two basal arthropods, the myriapod Strigamia maritima and the chelicerate Ixodes scapularis. In both, Dscam diversified extensively by whole gene duplications resulting in multigene expansions. Within some of the S. maritima genes, exons coding for one of the immunoglobulin domains (Ig7) duplicated and are mutually exclusively alternatively spliced. Our results suggest that Dscam diversification was selected independently in chelicerates, myriapods, and pancrustaceans and that the usage of Dscam diversity by immune cells evolved for the first time in basal arthropods. We propose an evolutionary scenario for the appearance of the highly variable Dscam gene of pancrustaceans, adding to the understanding of how alternative splicing, exon, and gene duplication contribute to create molecular diversity associated with potentially new cellular functions.     

Population genetics of duplicated alternatively spliced exons of the Dscam gene in Daphnia and Drosophila

In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam.     

Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.     

The organization and evolution of the dipteran and hymenopteran Down syndrome cell adhesion molecule (Dscam) genes

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor and can generate 38,016 different isoforms via the alternative splicing of 95 variable exons. Dscam contains 10 immunoglobulin (Ig), six Fibronectin type III, a transmembrane (TM), and cytoplasmic domains. The different Dscam isoforms vary in the amino acid sequence of three of the Ig domains and the TM domain. Here, we have compared the organization of the Dscam gene from three members of the Drosophila subgenus (D. melanogaster, D. pseudoobscura, and D. virilis), the mosquito Anopheles gambiae, and the honeybee Apis mellifera. Each of these organisms contains numerous alternative exons and can potentially synthesize tens of thousands of isoforms. Interestingly, most of the alternative exons in one species are more similar to one another than to the corresponding alternative exons in the other species. These observations provide strong evidence that many of the alternative exons have arisen by reiterative exon duplication and deletion events. In addition, these findings suggest that the expression of a large Dscam repertoire is more important for the development and function of the insect nervous system than the actual sequence of each isoform.     

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms

We have previously isolated and characterized cloned complementary DNAs (cDNAs) for striated and smooth muscle alpha-tropomyosin. The sequences of these cDNA clones suggested that these two isoforms were encoded by the same gene. Here, we have determined the complete structure of the alpha-tropomyosin (alpha-TM) gene, establishing that a single gene, with a sequence complexity of 28 kilobase pairs, is split into 12 exons and produces the smooth and striated muscle alpha-TM mRNA isoforms by alternative splicing of a minimum of five exchangeable isotype-specific exons. The elucidation of the intron/exon organization of alpha-TM suggests that this gene evolved from an ancestral gene encoding a 21-aa protein that might represent the primordial actin binding domain. Sequence comparison between the pairs of exons coding for the "isotype switch regions" and among the corresponding regions of tropomyosin genes in a variety of species ranging from insects to mammals, suggests that the alternatively spliced exons are very old and might have arisen before the radiation of the arthropods, more than 600 million years ago. Additionally, the examination of the intronic sequences has uncovered potential alternative intramolecular secondary structures (hairpin-loop structures) which might be involved in the tissue-specific expression of the duplicated and mutually exclusive alpha-TM isotype-specific exons.     

A regulator of Dscam mutually exclusive splicing fidelity

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.     

Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported

Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.     

Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures

Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons--the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA.     

Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.     

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.     

Self-recognition at the atomic level: understanding the astonishing molecular diversity of homophilic Dscams

The Drosophila Dscams are immunoglobulin superfamily members produced from a single gene that is diversified by alternative splicing to produce a family of cell-surface proteins with over 19,000 different ectodomain isoforms. Dscams are critical for neuronal wiring, and mounting evidence suggests that they play a key role in self-avoidance between sister branches from neurons, which depends on homophilic self-recognition by Dscams. Two recent papers shed new light on Dscam recognition: first by showing that the vast majority of Dscam isoforms mediate specific homophilic binding and second by revealing the essence of the molecular basis of homophilic recognition by Dscams through high-resolution structural studies.     

Wavelength-dependent polarization orientation in Daphnia

The ability to detect and use the polarization of light for orientation is widespread among invertebrates. Among terrestrial insects, the retinula cells that are responsible for polarization detection contain a single visual pigment, either ultraviolet or short (blue) wavelength sensitive. With the exception of a few aquatic insects, the visual pigments underlying polarization sensitivity in aquatic invertebrates have yet to be determined. Here we report that polarotaxis in Daphnia pulex, a freshwater crustacean, is wavelength dependent and most likely mediated by two visual pigments with absorbance maxima in the middle (green) and long wavelength (red) parts of the spectrum. This contrasts with the response of a closely related species, D. magna, in which polarotaxis is wavelength independent and based on a single middle wavelength visual pigment. The visual systems in Daphnia are the first among crustaceans shown to utilize a middle wavelength pigment for polarization detection and, in the case of D. pulex, the first shown to use more than one visual pigment for such a purpose.     

Spiny lobster development: mechanisms inducing metamorphosis to the puerulus: a review

This review outlines current knowledge of mechanisms effecting metamorphosis in decapod crustaceans and insects. The comparative approach demonstrates some of the complexities that need resolving to find an answer to the question raised frequently by ecologists: “What triggers metamorphosis in spiny lobsters?” It is evident that crustacean moulting and metamorphosis are genetically controlled through endocrine systems that mediate gene expression. The molecular mechanisms underlying these developmental processes have been studied intensively in insects, particularly in the fruitfly, Drosophila melanogaster (Diptera), and some lepidopteran species. Comparatively, there is minimal information available for a few decapod crustacean species, but none for spiny lobsters (Palinuridae). Nothing was known of hormone signalling transduction pathways, via nuclear receptors (NRs) and gene activation during larval moults in palinurids—until a recent, ground-breaking study of early phyllosomal development of Panulirus ornatus by Wilson et al. (Rock Lobster Enhancement and Aquaculture Subprogram. FRDC Project 2000/263, Australian Govt, Fisheries Research and Development Corporation and Australian Institute of Marine Science, Nov 2005). Their study not only identified homologues of five hormone NRs of D. melanogaster, but also patterns of gene regulation showing strong similarities to those of gene expression found in insect larval development. Their results indicated that control of moulting and metamorphosis in palinurids closely parallels that in insects, suggesting that insects can serve as model systems for elucidating molecular mechanisms in larval decapods. In insects and crustaceans, the steroid hormone, ecdysone, (20E) initiates moulting. In insects, juvenile hormone (JH) mediates the type of larval moult that occurs, either anamorphic or metamorphic. The latter results when the level of JH in the haemolymph drops in the final larval instar. High levels of JH inhibit the metamorphic moult during insect larval development. The interaction of 20E and JH is not fully understood, and the operative molecular mechanisms are still being elucidated. No nuclear receptor for JH has been identified, and alternative JH signalling pathways await identification. In decapod crustaceans, methyl farnesoate (MF), a precursor of JH, replaces the latter in other functions mediated by JH in insects; but there is little evidence indicating that MF plays a similar ‘antimetamorphic’ role in decapod larval moults.     

AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system

Activation of the insect innate immune system is dependent on a limited number of pattern recognition receptors (PRRs) capable of interacting with pathogen-associated molecular pattern. Here we report a novel role of an alternatively spliced hypervariable immunoglobulin domain-encoding gene, Dscam, in generating a broad range of PRRs implicated in immune defense in the malaria vector Anopheles gambiae. The mosquito Down syndrome cell adhesion molecule gene, AgDscam, has a complex genome organization with 101 exons that can produce over 31,000 potential alternative splice forms with different combinations of adhesive domains and interaction specificities. AgDscam responds to infection by producing pathogen challenge-specific splice form repertoires. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria and the malaria parasite Plasmodium. AgDscam is mediating phagocytosis of bacteria with which it can associate and defend against in a splice form–specific manner. AgDscam is a hypervariable PRR of the A. gambiae innate immune system.     

AgDscam, a Hypervariable Immunoglobulin Domain-Containing Receptor of the Anopheles gambiae Innate Immune System

Activation of the insect innate immune system is dependent on a limited number of pattern recognition receptors (PRRs) capable of interacting with pathogen-associated molecular pattern. Here we report a novel role of an alternatively spliced hypervariable immunoglobulin domain-encoding gene, Dscam, in generating a broad range of PRRs implicated in immune defense in the malaria vector Anopheles gambiae. The mosquito Down syndrome cell adhesion molecule gene, AgDscam, has a complex genome organization with 101 exons that can produce over 31,000 potential alternative splice forms with different combinations of adhesive domains and interaction specificities. AgDscam responds to infection by producing pathogen challenge-specific splice form repertoires. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria and the malaria parasite Plasmodium. AgDscam is mediating phagocytosis of bacteria with which it can associate and defend against in a splice form–specific manner. AgDscam is a hypervariable PRR of the A. gambiae innate immune system.     

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.     

Generalists as sequential specialists: diets and prey switching in juvenile silver perch

Diets of silver perch, Bidyanus bidyanus, in organically fertilised aquaculture ponds were dominated by chironomid larvae, Daphnia and calanoid copepods. Insects and crustaceans contributed approximately 80% and 20% by weight to the diet respectively. Classification of the stomach contents of individual fish revealed 8 diet groups, 4 of which were dominated by planktonic crustaceans and 4 by insects. Each diet group was strongly dominated by a different prey type. Fish from the same sample tended to belong to the same diet group and there was a non-random distribution of diet groups across ponds. Perch diets were influenced by the method of pond fertilisation (livestock effluent or pellet feed). Shifts in the representation of groups indicated that fish switched from one diet group to another over a 2–4 week period. The selection of planktonic prey by perch was related to prey densities in the ponds. Fish preferred Daphnia when these prey were common, but switched to calanoids and insects when Daphnia were scarce. A perfect rank correlation between the mean body size of planktonic prey and their contribution to the diet suggested that prey choice involved comparative decisions based on prey size. These findings indicate that, while classified as dietary generalists, silver perch exhibit consumption patterns which at the individual level are highly specialised at any given time. These patterns can be predicted, given information on prey densities in the environment.