Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity.

The Down syndrome cell adhesion molecule 1 (Dscam1) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for nervous and immune functions. Chelicerates generate approximately 50 to 100 shortened Dscam (sDscam) isoforms by alternative promoters, similar to mammalian protocadherins. Here, we reveal that trans-splicing markedly increases the repository of sDscamß isoforms in Tetranychus urticae. Unexpectedly, every variable exon cassette engages in trans-splicing with constant exons from another cluster. Moreover, we provide evidence that competing RNA pairing not only governs alternative cis-splicing but also facilitates trans-splicing. Trans-spliced sDscam isoforms mediate cell adhesion ability but exhibit the same homophilic binding specificity as their cis-spliced counterparts. Thus, we reveal a single sDscam locus that generates diverse adhesion molecules through cis- and trans-splicing coupled with alternative promoters. These findings expand understanding of the mechanism underlying molecular diversity and have implications for the molecular control of neuronal and/or immune specificity.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing architecture-driven splicing bias in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D-/- and DscamΔ9D-/-), respectively. In contrast to DscamΔ4D-/-, DscamΔ9D-/- exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.

Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition.

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. We report that Chelicerata sDscamα and some sDscamß protein trans interactions are strictly homophilic, and that the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. We report that cell-cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, our data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. We propose that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self-nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, our results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, our findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self-nonself discrimination.

A chelicerate-specific burst of nonclassical Dscam diversity.

BACKGROUND: The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. RESULTS: In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types-mDscam, sDscamα, sDscamß, and sDscamγ-based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5' cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. CONCLUSIONS: This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Role and convergent evolution of competing RNA secondary structures in mutually exclusive splicing.

Exon or cassette duplication is an important means of expanding protein and functional diversity through mutually exclusive splicing. However, the mechanistic basis of this process in non-arthropod species remains poorly understood. Here, we demonstrate that MRP1 genes underwent tandem exon duplication in Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, Echinodermata, and early-diverging Chordata but not in late-diverging vertebrates. Interestingly, these events were of independent origin in different phyla, suggesting convergent evolution of alternative splicing. Furthermore, we showed that multiple sets of clade-conserved RNA pairings evolved to guide species-specific mutually exclusive splicing in Arthropoda. Importantly, we also identified a similar structural code in MRP exon clusters of the annelid, Capitella teleta, and chordate, Branchiostoma belcheri, suggesting an evolutionarily conserved competing pairing-guided mechanism in bilaterians. Taken together, these data reveal the molecular determinants and RNA pairing-guided evolution of species-specific mutually exclusive splicing spanning more than 600 million years of bilaterian evolution. These findings have a significant impact on our understanding of the evolution of and mechanism underpinning isoform diversity and complex gene structure.

A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.