Competing RNA Pairings Regulate Cis-and Trans-splicing In The 5’ Variable Region Of S Dscam

Drosophila melanogaster Down syndrome cell adhesion molecule1(Dscam1)locus encodes potential 38,016 distinct isoforms via alternative splicing.This vast repertoire of Dscam1 molecules endows each neuron with a unique molecular identity,allowing it to distinguish between self and nonself.In vertebrates,the clustered protocadherins(cPcdhs)generate isoform diversity through variable promoters to perform similar functions.Recently,our group discovered the shortened Dscam(sDscam)in the subphylum Chelicerata,which has high sequence similarity with fly Dscam1,and shares a striking organizational resemblance to the 5’ variable regions of vertebrate cPcdh.However,compared with the well-characterized properties of fly Dscam1 and vertebrate cPcdh,the definitive molecular mechanisms that regulate sDscam isoform diversity are still poorly understood in Chelicerata.In this study,systematic experiments have been conducted on the isoform diversity regulatory mechanism of the Tetranychus urticae sDscamβ and its cell adhesion function.The results are as follows:Firstly,the results suggest that trans-splicing strikingly increases the repository of the sDscamβ isoforms,and competing RNA pairings not only govern cis-alternative splicing,but also facilitated trans-splicing.Analysis of RNA-seq data revealed that every variable exon cassette of the sDscamβ locus engages in trans-splicing with constant exons from another cluster,which significantly increased the number of isoforms.Through comparative sequence analysis,the base-pairing interaction between the docking site and selector sequence was identified in sDscamβ.Effects of competing RNA pairings were validated by disruptive single mutations and compensatory double mutations of RNA secondary structures.When RNA secondary structure was disrupted,the amount of adjacent 5′ variable exon cassette spliced into mature m RNA was significantly reduced,while when RNA secondary structure was restored by compensatory double mutations,the amount of 5′ variable exon cassette spliced into the mature m RNA was restored to the wild-type level.Sequence analysis found that the docking site could be paired with almost any selector sequence from another gene cluster.Disruption of the intergenic RNA secondary structure can significantly reduce the trans-spliced products,while restoration of the intergenic RNA secondary structure restored the trans-spliced products to the wild-type level.The results of this study show that intronic competing RNA pairings can not only regulate cis-alternative splicing,but also promote trans-alternative splicing.Based on the above series of experimental data,a model for the regulation of alternative cis-and trans-splicing by intronic competing RNA secondary structure was established.On this basis,the cell adhesion function of sDscamβ isoforms was further analyzed.Cell aggregation experiments showed that both cis-and trans-spliced sDscamβisoforms could mediate cell aggregation.However,the marked differences in aggregation ability among isoforms with the same variable region but different constant regions,suggest that the distinct constant domains generated by trans-splicing may affect the activity of homophilic trans-binding.The binding specificity assay showed that sDscamβ exhibit homophilic binding specificity.Swapping the variable Ig1 domain of a given sDscamβ for that of another led to a shift in binding specificity.By contrast,swapping the variable Ig2 domain or constant region of sDscamβ resulted in no change in specificity.These data indicate that the variable Ig1 domain of sDscamβ is necessary and sufficient to determine its adhesive specificity.Homology modeling showed that the variable Ig1 domain of sDscamβ interacted in an antiparallel orientation,similar to vertebrate cPcdh and Mesobuthus martensii sDscam.Moreover,co-expression of multiple sDscamβ isoforms with different N-variable domains generates new combinatorial homophilic specificity,whereas co-expression of multiple sDscamβisoforms with the same variable region but different constant regions fails to generate that.The results of this study show that trans-splicing mediated by RNA pairings expands sDscam isoform diversity but not homophilic binding specificity.Combined with the above series of experimental data,the mechanism model of sDscamβcombinatorial homophilic specificity was established.Taken together,this study reveals an extreme sDscamβ locus that generates highly diverse protein isoforms through alternative cis-and trans-splicing coupled with alternative promoters;establishes a model for the regulation of alternative cis-and trans-splicing by intronic competing RNA secondary structure.The binding specificity assay showed that trans-spliced sDscam isoforms mediate cell adhesion ability but exhibit the same homophilic binding specificity as their cis-spliced counterparts.These findings greatly expand our understanding of the mechanism regulating molecular diversity and have important implications for elucidating the molecular basis of neuronal specificity.