Drosophila Dscam Research Articles

A Vast Repertoire of Dscam Binding Specificities Arises from Modular Interactions of Variable Ig Domains

Dscam encodes a family of cell surface proteins required for establishing neural circuits in Drosophila. Alternative splicing of Drosophila Dscam can generate 19,008 distinct extracellular domains containing different combinations of three variable immunoglobulin domains. To test the binding properties of many Dscam isoforms, we developed a high-throughput ELISA-based binding assay. We provide evidence that 95% (>18,000) of Dscam isoforms exhibit striking isoform-specific homophilic binding. We demonstrate that each of the three variable domains binds to the same variable domain in an opposing isoform and identify the structural elements that mediate this self-binding of each domain. These studies demonstrate that self-binding domains can assemble in different combinations to generate an enormous family of homophilic binding proteins. We propose that this vast repertoire of Dscam recognition molecules is sufficient to provide each neuron with a unique identity and homotypic binding specificity, thereby allowing neuronal processes to distinguish between self and nonself.

Specific Drosophila Dscam Juxtamembrane Variants Control Dendritic Elaboration and Axonal Arborization

Drosophila Dscam isoforms are derived from two alternative transmembrane/juxtamembrane domains (TMs) in addition to thousands of ectodomain variants. Using a microRNA-based RNA interference technology, we selectively knocked down different subsets of Dscams containing either the exon 17.1- or exon 17.2-encoding TM. Eliminating Dscam[TM1] reduced Dscam expression but minimally affected postembryonic axonal morphogenesis. In contrast, depleting Dscam[TM2] blocked axon arborization. Further removal of Dscam[TM1] enhanced the loss-of-Dscam[TM2] axonal phenotypes. However, Dscam[TM1] primarily regulates dendritic development, as evidenced by the observations that removing Dscam[TM1] alone impeded elaboration of dendrites and that transgenic Dscam[TM1], but not Dscam[TM2], effectively rescued Dscam mutant dendritic phenotypes in mosaic organisms. These distinct Dscam functions can be attributed to the juxtamembrane regions of TMs that govern dendritic versus axonal targeting of Dscam as well. Together, we suggest that specific Drosophila Dscam juxtamembrane variants control dendritic elaboration and axonal arborization.

Alternative Splicing at NAGNAG Acceptors: Simply Noise or Noise and More?

Alternative splicing at pairs of acceptors in close proximity are one frequent cause of transcriptome complexity. In particular, acceptors with the pattern NAGNAG are widespread in several genomes [1–3]. When affecting the coding regions, alternative splicing at NAGNAGs mainly results in the insertion/deletion of one amino acid. While such subtle events are undoubtedly frequent, an important question arises: do they have functional consequences or are they simply noise tolerated by cells? Zavolan and colleagues [3,4] suggest that these variations are the result of stochastic binding of the spliceosome at neighboring splice sites and do not discuss known functional implications. We previously found indications against a general noise assumption for NAGNAG splice events [1]: biases towards intron phase 1 and single amino acid insertions/deletions, correlation of amino acid variation and the peptide environment, enrichment of polar residues at NAGNAG exon–exon junctions, preference for protein–protein interactions and particular Pfam domains, human–mouse conservation of the intronic AG, and tissue-specific splicing at several NAGNAG acceptors. These findings indicate negative selection against NAGNAG-derived variability deleterious for certain protein regions, which agrees with the underrepresentation of NAGNAGs in coding regions detected by Zavolan and colleagues [4]. This does not rule out that variability may be advantageous for other proteins, but signs of positive selection are much harder to detect and remain to be shown. Zavolan's finding that confirmed NAGNAGs (current mRNAs/expressed sequence tags do show alternative splicing) are not better conserved between human and mouse than unconfirmed ones may argue against functional implications. However, this result is probably biased by the unconfirmed dataset, which consists of ~60% NAGGAG whose GAG is part of the conserved exon. To avoid such a bias, we split confirmed NAGNAGs into those in which the “extra” AG is either intronic or exonic, according to the transcript annotation [1]. Interestingly, intronic but not exonic extra AGs have a significant conservation. Meanwhile, Akerman and Mandel-Gutfreund found a high conservation of the intronic flanking regions [5], typical for biologically meaningful alternative splicing [6]. The finding of Zavolan and colleagues that relative acceptor strength is predictive for confirmed and unconfirmed NAGNAGs refers to an accepted fact of splicing (for example, alternative exons have weaker splice sites than constitutive ones [7]). In tandems, the splice-site strength often determines the preferred acceptor, consistent with our earlier results (see Supplementary Notes in [1]). Thus, we agree that thermodynamic fluctuation plays an essential role during splice-site recognition at NAGNAG acceptors. This is in line with the finding that a single mutation is sufficient to convert a normal acceptor into a NAGNAG tandem, enabling alternative splicing [8]. However, this useful model is not valid for all NAGNAGs. In particular, tissue-specific regulation of alternative NAGNAG splicing challenges this model [1,9]. Overrepresented sequence motifs found in the vicinity of confirmed NAGNAGs are likely to contribute to this regulation [5]. Moreover, some protein isoforms derived by alternative splicing at NAGNAG acceptors are known to be functionally different: IGF1R, signaling [10]; DRPLA, cellular localization [9]; mouse Pax3, DNA binding [11]; and Arabidopsis thaliana U11-35K, protein binding [12]. Alternative NAGNAG splicing in the untranslated region of mouse Ggt1 affects the translational efficiency [13]. Furthermore, a NAGNAG mutation in ABCA4 is relevant for Stargardt disease 1 [14]. For clarity, we did not claim that all alternative splice events at NAGNAGs serve as protein “fine-tuning” mechanism [1,8] (as misinterpreted by [4]). In our opinion, like genetic variants, splice variants may be neutral or result in phenotypic differences. Thus, they represent just another playground of molecular evolution [15,16]. The few currently evident cases of biologically different NAGNAG-derived isoforms may represent just the tip of an iceberg. Finally, in the context of the problem discussed here, it has to be considered that noise is important for many biological processes [17], leading to the model of “cultivated noise” [18]. For example, splicing noise at the Drosophila Dscam gene is used for cell individualization [19]. Although it has yet to be proven, it is tempting to speculate that noise arising by splicing at NAGNAG acceptors provides another “cultivated” stochastic mechanism. In conclusion, it remains unknown what fraction of the more than 1,900 currently confirmed human NAGNAGs play a role in biological functions. To facilitate further experimental and bioinformatics analyses, we developed a database, TassDB (http://helios.informatik.uni-freiburg.de/TassDB), that provides information and large collections of NAGNAG acceptors.

Variable window binding for mutually exclusive alternative splicing

A mechanism called Variable Window Binding for mutually exclusive alternative splicing is described for exon 6 of the Drosophila Dscam gene, which represents an extreme case of alternative splicing.

Alternative Splicing— When Two’s a Crowd

Various mechanisms have been proposed to explain mutually exclusive splicing of pairs of exons. A paper in this issue of Cell (Graveley, 2005) provides a fascinating insight into the perplexing question of how only one exon at a time is chosen from an array of 48 exons in the Drosophila Dscam gene.

The Development of Neuronal Morphology in Insects

Neurons are highly polarized cells with some regions specified for information input--typically the dendrites--and others specialized for information output--the axons. By extending to a specific location and branching in a specific manner, the processes of neurons determine at a fundamental level how the nervous system is wired to produce behavior. Recent studies suggest that relatively small changes in neuronal morphology could conceivably contribute to striking behavioral distinctions between invertebrate species. We review recent data that begin to shed light on how neurons extend dendrites to their targets and acquire their particular branching morphologies, drawing primarily on data from genetic model organisms. We speculate about how and why the actions of these genes might facilitate the diversification of dendritic morphology.

Alternative Splicing of Drosophila Dscam Generates Axon Guidance Receptors that Exhibit Isoform-Specific Homophilic Binding

Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. We demonstrate that different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. We propose that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.

The Down Syndrome Cell Adhesion Molecule (DSCAM) Interacts with and Activates Pak

The Down syndrome cell adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily that maps to a Down syndrome region of chromosome 21q22.2-22.3. In Drosophila, Dscam functions as an axon guidance receptor regulating targeting and branching. Genetic and biochemical studies have shown that in Drosophila, Dscam activates Pak1 via the Dock adaptor molecule. The extracellular domain of human DSCAM is highly homologous to the Drosophila protein; however, the intracellular domains of both human and Drosophila DSCAM share no obvious sequence identity. To study the signaling mechanisms of human DSCAM, we investigated the interaction between DSCAM and potential downstream molecules. We found that DSCAM directly binds to Pak1 and stimulates Pak1 phosphorylation and activity, unlike Drosophila where an adaptor protein Dock mediates the interaction between Dscam and Pak1. We also observed that DSCAM activates both JNK and p38 MAP kinases. Furthermore, expression of the cytoplasmic domain of DSCAM induces a morphological change in cultured cells that is JNK-dependent. These observations suggest that human DSCAM also signals through Pak1 and may function in axon guidance similar to the Drosophila Dscam.

Drosophila Dscam Is Required for Divergent Segregation of Sister Branches and Suppresses Ectopic Bifurcation of Axons

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.

Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing.

Here we investigate the promoter control of alternative splicing by studying two transcriptional activators on templates under replicating conditions. SV40 large T-antigen (T-Ag) activates template replication only 2-fold but transcription 25-fold. T-Ag-mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10- to 30-fold increase in the inclusion of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T-Ag and VP16 promote conspicuous co-localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co-localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.

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.