RNA Secondary Structure In Mutually Exclusive Splicing Of Dscam And Serpent Pre-mRNA

Alternative splicing is an important way of gene post-transcriptional processing, which plays a major role in the generation of proteomic and functional diversity in metazoan organisms. The Down’s syndrome cell adhesion molecule (Dscam) is the textbook example of alternative splicing, which could potentially encode 38,016 distinct cell-surface protein isoforms of the immunoglobulin superfamily in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster.Comparative genomics analysis indicates the intervening intron between exon 17.1 and 17.2 is below 57 nt in all insect species investigated, except for Diptera. Subsequently, our results reveale that the distance between branch point just before exon 17.2 and 5’splice site of exon 17.1 is lower than threshold (i.e.,50-60 nt in mammalian and Drosophila) in flies and mosquitoes. Therefore, this mutually exclusive behavior is enforced by steric hindrance between U1 and U2 snRNP while they combined with the 5’splice site and branch point respectively. Intron-exon RNA structures are evolutionarily conserved in 38 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulate the selection of exon 17 variants via masking 3’splice site. Moreover, there are two pairs of specific RNA structures in Drosophila, a previously undiscovered RNA pairing specifically activates exon 17.1 by bringing splice sites closer together, while the other functions moderately to hinder the accessibility of polypyrimidine sequences by splicing factors (i.e., U2AF65). Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution, provide a model of the regulation of alternative splicing through steric hindrance in combination with dynamic RNA secondary structure.In order to verify the universality of the above regulation model, some genes characterized by mutually exclusive splicing have been investigated. Excitedly, we reveal a novel melecular model regulating the mutually exclusive splicing of serpent pre-mRNA based on the competition between bidirectional RNA pairings. Comparative genomics analysis indicate four evolutionarily conserved intronic elements in Drosophila serpent exon 4, which potentially form upstream and dowestream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exon and facilitate mutually exclusive splicing fidelity. Upstream RNA pairing directs the inclusion of exon 4.1, while downstream RNA pairing activates exon 4.2 inclusion frequency. The selection of exon 4 largely depends on the relative thermodynamic stability between upstream and downstream RNA pairings and splice site strength. Importantly, the heritable expansion of bidirectional RNA pairings could guarantee mutually exclusive splcing in an ever-increasing variable exons, and facilitate the genetic evolution of organisms compared with a previously single docking-selector RNA pairing model. Moreover, similar bidirectional architectures were also found in Dscam exon 4,9 and RIC-3. We then propose a novel bidirectional competing pairing model of commitment to a single choice from a tandem array of multiple exons, and may provide new insight into the function of long-range RNA-RNA interactions in gene regulatory networks.