Investigation On Evolutionary Dynamics And Cis Elements Of Mutually Exclusive Splicing Of Caenorhabditis MRP1Gene And Hymenoptera Mhc Gene

Alternative Splicing is an essential processing mechanism after RNA transcription, and it widespreadly exists in eukaryotes for gene regulation and generating proteinic diversity. In several different types of alternative splicing, we focus on the mutually exclusive splicing, which only one exon is chosen out of a cluster of alternative exons arranged in a tandem array in mature mRNAs. by choosing different alternative exons to form different mature mRNA, mutually exclusive splicing can increase proteinic diversity. So far, not so many researches have been done to study MRP1and Mhc,in which exist mutually exclusive splicing.As one member of the ATP-binding cassette transmembrane transporter superfamily, the multidrug-resistance associated protein which encoded by MRP is an important factor involved in multidrug resistance of tumor cells. In Caenorhabditis elegans, we find that there are4mutually exclusive exons of MRP1gene, the results of RT-PCR and enzyme digestion experiment shows Exon2and Exon3are mainly included. In our study we analyze MRP1orthologous sequences of9Caenorhabditis, and results tell that there might be some cis elements in the introns of MRP1. Then we use bioinformatics methods such as BLAST to acquire the orthologous gene sequences from115various species including C.elegans in different gene databases. And with comparative genomic analyses of mutually exclusive splicing, we analyze evolutionary dynamics of MRP1splice variants and find it changes a lot in different species, and it also means these splice exons are species-specific and lineage-specific. Next step, we construct wild-type MRP1minigene expression vector of Caenorhabditis elegans, and microinject it into Caenorhabditis elegans in vivo. The result shows that the F2generations of Caenorhabditis elegans behave strangely and move as a circle.This phenomenon means rol-6markers works and our microinjection experiment is successful. Thus we preliminarily explore the conditions of MRP1microinjection experiment in studying mutually exclusive splicing mechanism. All above, these preliminary experimental data and technique would provide a strong basis for further study on the mutually exclusive splicing regulation mechanism of MRP1.Mhc gene can encode myosin heavy chain which is important part of myosin. MHC can expess some kind of different isofoms in skeletal muscle. In our study, we use BLAST to acquire the orthologous gene sequences from12Hymenoptera species. Evolutionary dynamics shows that in5clusters of mutually exclusive exons of Mhc,4of which are conserved except the third cluster which are dynamic. Comparative genomic analyses result shows that in the second cluster of Mhc, there are some conserved regions which may be cis elements. These preliminary experimental data and technique would provide a strong basis for further study on the mutually exclusive splicing regulation mechanism of Mhc.