Investigation On 3’ Mutually Exclusive Splicing Regulation Mechanism Of Drosophila CG30427 Pre-mRNAs

Alternative splicing is an important processing mechanism of eukaryotes precursor mRNA,which is conducive to increase protein diversity and regulate gene expression level and tissue specificity. Mutually exclusive splicing is an important form of alternative splicing. In previous studies of mutually exclusive splicing, two(or more)splicing events are not independent anymore,but are executed or disabled in a coordinated manner,only one variable exon is chosen to be cut into the mature mRNA isoform.However, studies about multiple exons as a whole to participate in the mutually exclusive splicing are very rare.CG30427 genes is a protein-coding genes, its coding protein has fatty acyl coenzyme A reductase activity.It is involved in the biological process such as: determination of adult lifespan; phagocytosis. Drosophila melanogaster CG30427 gene has 2 constitutive exons and 3 multi-exon clusters,which contain 4 exons,4 exons and 5 exons. The 3 multi-exon clusters exist mutually exclusive splicing, only one multi-exon cluster cut into mature mRNA finally.In this study, We analysized CG30427 gene sequences of 13 species of Drosophila through bioinformatics method,found introns within variable multi-exon clusters are smaller and have strong splicing signals. We designed experiments to extend the introns within variable multi-exon clusters and abate splicing signal strength.The experimental results shows exons no longer spliced together as a whole when introns within variable multi-exon clusters were extended or splicing signal were abated. In addition, we also proved that the introns within multi-exon clusters spliced preferentially.We also found 2 conservative sequence upstream variable multi-exon clusters 1 and 2 in fruit flies CG30427 gene by comparative genomics.Deletion mutation experiments shows these two conservative sequence influence the regulation of mutually exclusive splicing in D.melanogaster CG30427 gene.To sum up, the study reveals the reasons and mechanism of multiple exons as a whole to participate in the mutually exclusive splicing, enrich the connotation of mutually exclusive splicing; In addition, we find two conservative sequence influence CG30427 gene’s regulation of mutually exclusive splicing,which laid a foundation for further research.