唐氏综合症细胞粘附分子（Dscam）mRNA 互斥可变剪接调控机制的研究

Alternative splicing is a crucial mechanism for generating proteomic diversity. The Down syndrome cell adhesion molecule (Dscam) gene encoding 38,016 distinct isoforms via extensive alternative splicing, has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam was introduced repeatedly in many articles and books, but its regulation mechanisms of alternative splicing remained unclear. Our previous studies revealed that the noncoding sequence elements of Dscam pre-mRNA might play important roles on regulation of alternative splicing. In the current study, we decide to uncover its mechanisms of alternative splicing regulation. Firstly, the functions of noncoding sequence elements in alternative splicing regulation will be investigated by analysis of imformatics and RNA compensatory mutagenesis. Secondly, the unique regulators of alternative splicing will be identified, and how do the RNA sequence elements react with protein factors for regulation splicing of alternative exons will also be investigated . Because the Dscam is required in nervous system, so this study may provide not only new clues for investigating the mechanisms for gene expression regulation, but also therapeutic targets for some neural diseases.

果蝇的Dscam是一个与神经和免疫功能相关的基因，该基因通过可变剪接能产生38,016种成熟mRNA，但其调控机制仍不清楚。我们前期的研究发现Dscam 前体mRNA中的非编码序列元件对可变剪接有调控作用（Nat Struct Mol Biol, 2011, 18:159-168,申请人为并列第一作者）。本项目在前期的工作基础上，旨在探明Dscam 可变外显子剪接的调控机制，研究工作拟从两方面展开：一方面通过生物信息学分析和RNA补偿突变等实验，探明前体mRNA 中的非编码序列元件对可变剪接的指导作用；另一方面筛选出特异性调控Dscam 可变外显子剪接的蛋白质，并探明RNA序列元件是如何与调控蛋白相互作用，从而启动并控制可变外显子的剪接。由于Dscam 与神经性功能相关，因此本项目的研究不仅为基因表达的调控机制的阐析提供新思路，并且有可能为神经性疾病的治疗提供候选靶点。

本项目鉴定筛选出了一些能特异性调控Dscam可变外显子剪接的蛋白，进行了RNA和调控蛋白结合的实验，揭示了RNA 解旋酶等一些RNA结合蛋白在该可变剪接调控中起重要作用；阐明了PGRP-LC基因可变剪接的调控机制：即竞争性RNA互补配对和反式因子共同调控PGRP-LC互斥可变剪接；发现黑腹果蝇的vulcan外显子2上鉴定了新产生的内含子（Intron L），Intron L与内含子保留相关，为内含子保留的可变剪接研究提供基础；发现 Cu/Zn-Sod exon 1 的3' 端可变剪接产生一种新Cu/Zn-Sod，研究表明是内含子序列的“外显子化”机制产生，是剪接位点和顺式作用元件之间存在共进化。部分研究工作已发表在Genome等杂志上。