Evolutionary And Functional Analysis On Alternative Splicing And RNA Editing Event In Synaptotagmin Ⅰ Gene

Maturation of eukaryotic RNA usually depends on rather delicate and complex post-transcriptional processes, including residual modification, isoformation, addition, editing, splicing and so on. Alternative splicing takes active roles in proteome extension, as well as conferring novel functions to macromolecules. Various types of alternative splicing patterns on regulatory aspect have drawn more and more attentions from scientists, especially their impact on tiny functional tuning through changing regional structure of biomolecules.Neuronal system is one of the most complicated one not only in anatomic level, but also in molecular level. The abundance of functional molecules and the complexity of regulatory network, bring about both robustness and efficiency. Post-transcriptional processing is also of great significance to normal function of this system.Base on previous discovery of alternative splicing phenomena in the synaptotagmin I gene from Aplysia californica, this thesis tries to take a broader view on alternative splicing events of invertebrate synaptotagmin I orthologs from evolutionary aspect using holometabolous insects as materials. In addition to the previous alternative splicing site (siteA) with two 3' splicing acceptor choices, a novel and relative more conserved throughout speices alternative splicing site (siteB) was identified. Simultaneously, another alternative splicing pattern was identified within Culex pipiens synatptotagmin I gene (siteA) where the distal 3' splicing acceptor is lost, using a decoy 5' donor site inside former exon. Thus we propose an idea that there might be a compensatory system underlies this regulation hotspot, where one splicing site loss may cause another splicing site gain of function.Within the relatively more conserved alternative splicing site (siteB), synaptotagmin I gene from Apis mellifera was the exception, as the distal 3' spicing acceptor site was lost through nucleic acid transition: a genomically encoded A takes the place of G, while in other species the corresponding nucleic acid is G. Thus alternative splicing at siteB is lost. To test this site's conservancy in evolutionary angle, reversal mutation was performed over minigene using site-directed mutagenesis, through which, the original A is replaced by G, thus an additional 3' spicing acceptor is created. Based on in vivo splicing experiments, the phenomenon of alternative splicing was rediscovered in the mutant minigene. This procedure proves that SiteB is surely conserved through evolution process, and exceptions are additional splicing site losses due to silent mutation.Homology modeling evidence provided that alternative splicing event at site B may result in an extra alpha-helix structure within C2B domain linker region. We may say that the presence and absence of tri-peptide are sure to differ in function.Further we investigated whether RNA editing has some effect on the Syt I C2B domain, and trypsin parital digestion result showed that RNA editing may have confer more stability to overall protein.