Dscam基因外显子簇6互斥可变剪接顺式元件LCR及相关反式因子的功能研究

Alternative splicing is an important way of increasing protein diversity. Dscam gene is one classic case in research of alternative splicing, which can produce 38,016 different transcription by mutually exclusive alternative splicing,but its molecular mechanism is still unclear. Experiments have indicated that Dscam exon clusters 6 regulated mutually exclusive splicing by RNA secondary structure. And, we also found the cis-element LCR can activity exon 6 splicing. Based on this finding, we will investigate the function of different subunits in LCR and screen the core elements by RNA deletion experiments, identify trans-factors interaction with core elements by RNA pull down and LC-MS/MS, study the mechanism of the interaction between trans-factors and core elements by EMSA and RNA-CLIP-Seq. And then, we will use the Drosophila melanogaster knock-out mutants, RNAi and overexpression analysis to study the regulatory function of trans-factors in mutually exclusive splicing and related phenotypes, and to understand the molecular mechanism of cis-element LCR and trans-factors that affect mutually exclusive splicing events. It will be of great value to elucidate the regulatory mechanism of RNA secondary structure mediated mutually exclusive splicing.

可变剪接是增加蛋白质多样性的重要方式，Dscam基因是可变剪接研究中的经典案例，通过互斥可变剪接可以产生38,016 种不同的转录本,但其分子机制尚不清楚。我们的前期实验表明Dscam基因外显子簇6是受RNA二级结构介导的互斥可变剪接机制调控，同时还发现了激活外显子6剪接的顺式元件LCR。在此基础上，我们将通过RNA缺失突变等实验对顺式元件LCR不同亚基的调控功能进行研究，甑别其中的核心元件；利用RNA pull down和LC-MS/MS技术，鉴定与核心元件作用的反式因子；采用EMSA和RNA-CLIP-Seq等技术，研究反式因子与核心元件作用的分子机制。进而，利用果蝇基因敲除突变体、RNAi和过度表达技术，研究反式因子在互斥可变剪接和相关性状中的调控作用。从而，探明顺式元件LCR及反式因子影响互斥可变剪接的分子机理，对阐明RNA二级结构介导的互斥可变剪接调控机制具有重要价值。

可变剪接是增加蛋白质多样性的重要方式，Dscam基因是可变剪接研究中的经典案例，通过互斥可变剪接可以产生38,016 种不同的转录本，但其分子机制尚不清楚。本项目通过缺失突变等实验分析了顺式元件LCR不同亚基的调控功能，并发现了其核心元件；同时利用RNA pull down和LC-MS/MS 技术，鉴定了与核心元件作用的反式因子；并利用RNAi和过度表达技术证明了反式因子在互斥可变剪接中的调控作用。该研究使我们对RNA二级结构介导的互斥可变剪接调控机制有了进一步的认识。同时，通过比较基因组分析和突变实验，我们发现了双端远距离RNA配对在互斥可变剪接中的作用及机理。