The Role Of RNA Secondary Structure In Dscam Exon4and Exon6Mutually Exclusive Splicing In

Abstract:RNA alternative splicing is an important part of the expression and regulation of gene transcription, widely exist in eukaryotes, and it can greatly increase the molecular diversity and organismal complexity. RNA mutually exclusive splicing (MES) is the most complex type of it, but its mechanism is still unclear. MES results in inclusion of only one exon from two or more continuously arranged variable exonsRNA MES has been found very early, but until now, its regulation mechanisms are still poorly understood. Drosophila melanogaster (D. melanogaster) Dscam gene is the most famous MES gene. It contains four groups of mutually exclusive exons, and can produce38,016kinds of different isotypes by MES. There are a variety of hypotheses for RNA MES mechanism, and Gravely proposed a docking site-selector sequence model by bioinformatics methods to explain mutually exclusive events of Dscam cluster6in2005, however, this mechanism is not suitable in other mutually exclusive exons, and cannot explain how this gene produce so many forms of alternative exons.Thus, on the basis of our laboratory preliminary work, we use bioinformatics methods to in-depth analyze the Dscam gene mutually exclusive exon4of Drosophila12species. The results show that some conserved sequence elements present in the exon4introns, and they are likely to Gravely’s docking site-selector sequence model. The selector sequence is located within introns behind each mutually exclusive exon, and the docking site located in the last intron. Moreover, the docking site and selector sequence are reversely complementary, with possibility of forming stem-loops RNA secondary structure. The selector sequences compete to pair the docking site, thus guiding exon4MES. These conserved selector sequences and docking site exhibit amazing conservative RNA secondary structure in different Drosophila species, suggesting that these conserved sequence elements possibly are key regulator to guide the MES of Dscam exon4. Subsequently series of experimental data confirm our conjecture of these specific sequence features.In addition, with bioinformatics methods, we find that there is a complex intron sequence in front of the docking site of Dscam gene exon6in20Drosophila species. It can form a compact6RNA stem-loop structures. Similar RNA secondary structures are evolutionarily conserved in the insect species. We infer that these intron sequences are an important component of the regulation of Drosophila Dscam exon6MES. We use Drosophila immortalized S2cell line to detect the Dscam gene exon6expression, carry out a series of experimental studies:by destroying the pairing secondary structure, complementary mutation of secondary structure, insertion or deletion of some of the conserved sequence. The experimental data show that:it may be the formation of six stem-loop structure of RNA pairing intron sequences that involved in MES of Drosophila Dscam exon6, the conservative RNA secondary structure plays a very important role in the MES.Further, by EMSA technique, we analyze the RNA binding protein binding capacity with the mutually exclusive exon and its intron sequence of Drosophila14-3-3C gene, and the enhancer-like sequence in front of the Drosophila Dscam exon6. Results show that the exon tends to combine with SR protein, intron sequence tends to combine with hnRNP, enhancer-like sequence tends to combine with SR protein.In summary, our studies on Dscam mutually exclusive exon4and6of model organism Drosophila melanogaster develop the hypothesis that Gravely have made on RNA mutually exclusive splicing, and reveal that the RNA secondary structure extensively exists in mutually exclusive splicing regulation.