Identification Of Related Cis Elements And RNA Secondary Structure Of Alternative Splicing And Evolutionary Conservation Analysis

Alternative splicing (AS) results in the generation of different isoforms and protein diversity from a single gene. Mutually exclusive splicing is one of the special form of AS. The most striking example in Drosophila melanogaster (D.me) is Dowm syndrome cell adhesion molecule (Dscam) mutually exclusive splicing. Drosophila melanogaster Dscam can generate 38,016 different isoforms through mutually exclusive splicing. MRP gene in Drosophila melanogaster also exists mutually exclusive splicing event, which can generate 16 differennt isoforms by mutually exclusive alternative splicing. The current research on mutually exclusive splicing model is only limited to the predicted model. Mutually exclusive splicing model is rarely reported in the related document. So far, mutually exclusive splicing mechanism of MRP gene and other related gene are still poorly understood.RNA secondary structures which on the competing base-pairing interactions between docking site and selection sequences are thought to inhibited mutually exclusive splicing exon in the loop and via shortening the effective intron distance to activate mutually exclusive splicing. However, the regulatory mechanism remain obscure due to complexity of selection and activation in mutually exclusive splicing exons. In our study, we selected representative MRP gene as the object of alternative splicing mechanism. We found two cases in Drosophila melanogaster MRP gene cluster 8 by using bioformatics analysis. One case is 7 mutually exclusive splicing exons in exon cluster 8 of MRP gene. The anther case is 8 mutually exclusive splicing exons in exon cluster 8 of MRP gene. We found single RNA pairing in exon cluster 8 of MRP gene which includes 7 mutually exclusive splicing exons and dual RNA pairings in exon cluster 8 of MRP gene which includes 8 mutually exclusive splicing exons. Exon cluster 8 of MRP gene which includes 8 mutually exclusive splicing exons have one more RNA secondry structure while compared with exon cluster 8 of MRP gene which includes 7 mutually exclusive splicing exons. The experiments proved that RNA secondary structure exists in exon cluster 8 of MRP gene. We also verified that RNA secondary structure of mutually exclusive splicing exons in exon cluster 8 of MRP gene can regulate the alternative splicing.Three genes were found the event of RNA secondary structures by using the method of bioformatics analysis which includes Nc73EF gene (CG11661), Srp gene (CG3992) and Eb1 gene (CG3265) in our study. Srp gene (CG3992) is the example of dual RNA pairing in alternative splicing. The discovery of dual RNA pairing in Srp gene (CG3992) may provide new clues for further revealing of the alternative splicing mechanism, and also may suggest a new alternative splicing regulation model.Dscam gene cluster 4, cluster 6 and cluster 9 were also do comparative genomic and phylogenetic analysis in Lepidoptera, Hymenoptera, Coleoptera and Crustacea species. Our results reveale that Exon cluster 4, cluster 6, cluster 9 of Dscam gene in Lepidoptera, Hymenoptera, Coleoptera, Crustacea have evolutionary conservation of RNA secondary structure. The informations provide a foundation for further study of Dscam gene alternative splicing mechanism.In order to further study alternative splicing mediated by RNA secondary structure at the level of protein, we conducted an evolutionary analysis of Drosophila melanogaster protein interaction network in model organism and predicted the protein interaction network in Stegodyphus mimosarum which involved in RNA splicing. Our results indicate that there is evolutionary conservation of the protein interaction network of RNA splicing between Drosophila melanogaster, homo sapiens and Arabidopsis thaliana.19 RNA splicing proteins were predicted from protein interaction network and the RNA splicing protein interaction network was constructed in S.mimosarum. These results provide some clues for the study of the protein internactions of mutually exclusive splicing, and provide a favorable basis for the complex mutually exclusive splicing mediated by RNA sencondary structure.