REGULATED SPLICING OF MESSENGER RNA PRECURSORS

这个项目的长期目标是了解这种机制。 通过其调节组织特异性剪接事件。这 需要阐明顺式作用序列和扩散 牵涉到细胞因素。为实现这些目标而采取的方法 目的是研究前速激肽原基因的剪接作为一种 组织特定剪接事件的模型。这是最简单的 可用于这些研究的模型。这项提案的重点是 导致跳跃的明显受调控的剪接事件 在大脑中优先表达倒数第二个3‘外显子，而不是 甲状腺组织。具体地说，此应用程序建议使用 遵循三个目标。(1)建立组织培养模型体系 用于研究细胞系中速激肽原原基因的表达 模仿这种基因的差异表达方式 在大脑和甲状腺组织中。(2)的顺序要求 这一外显子跳过事件将由Directed 基因区域序列的诱变研究 可选外显子。这将包括删除插入序列 以及剪接位点区的突变。RNA稳定性的作用 和核运输也将在研究中使用这些 细胞系来确定这种剪接事件的程度 指导不同细胞质的生产 速激肽原前体mRNA形成。(3)所需的细胞因素 将通过使用可溶性细胞的生化研究来阐明 提取前mRNA底物并对其建模 从测试的突变研究中获得的信息 培养的细胞系。理解这种组织的基础是- 生化研究将提供特定的剪接事件 来测试单个剪接位点的内在效率 剪接位点的上下文来自特征良好的预剪接位点 MRNAs。对受调控的组织特异性剪接的理解 事件对于全面理解发展是至关重要的 过程和组织特有的基因调控。

The long term goal of this project is to understand the mechanism by which a tissue-specific splicing event is regulated. This requires elucidation of both cis-acting sequences and diffusible cellular factors involved. The approach taken to achieve these goals is to study the splicing of the preprotachykinin gene as a model for a tissue-specific splicing event. This is the simplest model available for these studies. The focus of this proposal is the apparently regulated splicing event that results in the skipping of the penultimate 3' exon preferentially in brain, in contrast to thyroid tissue. Specifically, this application proposes the following three aims. (1) A tissue culture model system will be developed to study preprotachykinin gene expression in cell lines that mimic the way in which this gene is differentially expressed in brain and thyroid tissues. (2) The sequence requirements for this exon-skipping event will be elucidated by directed mutagenesis of sequences in the region of the gene containing the optional exon. This will include deletion of intervening sequences and mutagenesis of splice site regions. The role of RNA stability and nuclear transport will also be addressed in studies using these cell lines to determine the extent to which this splicing event directs the production of the different cytoplasmic preprotachykinin mRNA forms. (3) The required cellular factors will be elucidated by biochemical studies using soluble cellular extracts and model pre-mRNA substrates constructed using the information gained from the mutational studies tested in the cultured cell lines. The foundation for understanding this tissue- specific splicing event will be provided by biochemical studies that test the intrinsic efficiency of individual splice sites within the context of splice sites derived from well characterized pre- mRNAs. The understanding of a regulated, tissue specific splicing event is critical for a complete understanding of developmental processes and tissue-specific gene regulation.