Mutually Exclusive Alternative Splicing Mechanism Of Drosophila Dscam And Tep2

Alternative splicing (AS) results in the generation of different iso forms from a single gene by using different promoters, terminators or splicing sites. AS is a major mechanism for the enhancement of transcriptome and proteome diversity, and mutually exclusive alternative splicing is one of the special form of AS. The most striking example in Drosophila melanogaster (D.me) is observed in the Down syndrome cell adhesion molecule (Dscam), which can generate38,016different iso forms by mutually exclusive alternative splicing. Another extraordinary example is thioester-containing proteins2(Tep2) in D.me, which can be transcribed into only five iso forms by alternative splicing of exon5cluster but changes with a most fastest way during the evolutionary process. So far, the molecular mechanism of the AS still needs further discussion.RNA secondary structures, based on the competing base-pairing interactions between docking site and selection sequences, are thought to direct the mutually exclusive splicing of Dscam, but the underlying mechanisms are poorly understood. In this study, we describe a locus control region(LCR) upstream the docking site in Dscam exon6cluster which is comprised of tandem multi-’subunit’RNA structures and widely exists in multiple insect species. We also proved that LCR can activate the Dscam exon6cluster and specifically allow for the selection of only one exon variant via shortening the effective intron distance in combination with docking site-selector sequence interactions. Besides, we found some potential binding sites of regulators near the docking site. Our findings not only provide a locus control region-dependent mechanism for mutually exclusive splicing, but also suggest a model for the evolution of increased complexity in a long-range RNA molecular machine.Inspired by the discoveries in the Dscam exon6cluster, we then studied the alternative splicing mechanism of D.me Tep2exon5cluster. We find that the approximation-activating mechanism also fits for the alternative splicing of Tep2exon5cluster. We further proved two intron cis-acting elements that played an important role in the splicing of the Tep2exon5cluster. We then screened six proteins by RNA-pulldown and RNA interference experiments and put forward a hypothetical model for D.me Tep2mutually splicing, which infer that the activation of mutually exclusive splicing of the D.me Tep2exon5cluster via the approximation to constitutive exon6by competitive protein-protein interaction rather than direct RNA secondary structures. Our studies provide new clues to reveal the alternative splicing mechanism of Tep2exon5cluster, and also suggest a new molecular regulatory mechanism of mutually alternative splicing.