Investigation On Regulatory Proteins Of Alternative Splicing Of Drosophila14-3-3ζGene By RNAi

Splicing is an essential processing mechanism after RNA transcription. Alternative splicing is a major contributor to proteomic diversity by seven different patterns. Mutually exclusive splicing which only one exon is chosen out of a cluster of alternative exons arranged in a tandem array in mature mRNAs is one pattern of alternative splicing. It can increase proteomic diversity by choosing different alternative exons to form different muture mRNA. Many works have done to study the mechanism underlies the mutually exclusive splicing, but there still some problems are unclear.14-3-3gene family is conserved regulatory molecules expressed in all eukaryotic cells and paticipitated in many important pathways.14-3-3ζ gene including three alternative exons is a member of14-3-3gene family. Our previous studies of the regulation mechanism of14-3-3ζgene of Drosophila melanogaster have found the secondary structure in mutually exclusive splicing, in order to futher improve the mechanism, we established an RNA interference system in S2cell line of Drosophila melanogaster. We transfected S2cells with dsRNA and detected the effect of transfection by Semiquantitative RT-PCR. The main factors affecting the interference effect was found:the amount of double-stranded RNA, transfection time and the transfection frequency. Additonally, transfected with20μg dsRNA for3days has more than70%interference effect through gradient experiments, which provide a better platform for follow-up study. Investigation on regulatory proteins of alternative splicing of Drosophila14-3-3ζ gene by this RNAi system indicated that there were abnormal situation in which two mutually exclusive exons connected after Interfering regulatory protein Hrp36, Hrp38, Hrp48, SC35and Rbpl-like, but interfering with other proteins is not the case, so this five protein may play an important role in the14-3-3ζ gene alternative splicing regulation, and it laid a solid foundation for the14-3-3ζ genes mutually exclusive splicing regulation mechanism.