Mechanism And Function Of RNA Secondary Structure In A-I RNA Editing And Mutually Exclusive Splicing

RNA editing and alternative splicing are two important post-transcriptional processings leading to the increase of protein and functional diversity. Both are closely related to cell differentiation and development, the development of the disease and so on. The mechanism of A-I RNA editing has been elucidated. Adenosines (A) at the specific sites of double-stranded RNA were deaminated into inosine (I) by the ADARs. But its biological significance still has large controversy:increase protein diversity or maintain phylogenetic conservation? Drosophila melanogaster Dscam gene generates38,016different isoforms through mutually exclusive splicing. It’s a classic case of alternative splicing in many textbooks and monographs, but its mechanism is still poorly understood.Based on the analysis of the A-I RNA editing data in the Kv2K+channel from different insects spanning more than300million years of evolution:Drosophila melanogaster, Culex pipiens, Pulex irritans, Bombyx mori, Tribolium castaneum, Apis mellifera, Pediculus humanus and Myzus persicae, we found that the A-I RNA editing mostly altered less-conserved coding positions of those highly conserved coding regions. It reveals that RNA editing might play dual roles in evolution by extending protein diversity and maintaining phylogenetic conservation.In addition, we found the relationship between the nucleus A-I RNA editing and the DNA mutation by linking RNA editing and DNA mutation. The G to A mutation in the DNA level can be fixed by A-I RNA editing in mRNA after transcription. While edited I (G) can be firmly embedded into genomic DNA, leading an A to G mutation.Inspired by the RNA secondary structure of A-I RNA editing, we established a mutually exclusive splicing model system for studying its mechanism by RNA compensatory mutation. A class of cis-elements were found in Drosophila melanogaster14-3-3(?) gene, which regulated mutually exclusive splicing. These elements were species-specific and secondary structure conserved. Selection of mutually exclusive exons were regulated by competitive complementary pairing mechanism of these class-specific cis-elements in precursor mRNA.Further studies have shown that such cis-elements regulate the selection of mutually exclusive exons by a combinatorial mechanism. This mechanism includes close-to-activating mechanism, loopping-out mechanism and other cis-elements regulation. In addition, this study also shown that the selection frequency of mutually exclusive exons was closely related to small changes of RNA secondary structure. It was positively correlated with its RNA pairing strength, and was negatively correlated with the distance of the elements. This indicated selection of mutually exclusive exons was closely related to physical competition of RNA secondary structure. On this basis, the study shown RNA pairing mediated mutually exclusive splicing had the potentially evolutionary expansion ability by exon copy simulation experiments. At last, RNA complementary pairing structures of Drosophila melanogaster Dscam gene exon clusters4and9and MHC gene mutually exclusive exon7,9and11were decoded by according to the characteristics of the model:selector sequence and docking site conserved sequence located in introns, which are after mutually exclusive exons. Finally we elucidate the mechanism of mutually exclusive splicing in D. melanogaster Dscam clusters4and9, MHC mutually exclusive exons7,9and11and14-3-3(?) mutually exclusive exon5.