REGULATED SPLICING OF MESSENGER RNA PRECURSORS

本项目的长期目标是了解 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可扩散性 细胞因素参与。 为实现这些目标而采取的办法 目的是研究前速激肽原基因的剪接， 组织特异性剪接事件的模型。 这是最简单的 模型可用于这些研究。 这项建议的重点是 导致跳跃的明显受调控的剪接事件 的倒数第二个3'外显子优先在大脑中，相反， 甲状腺组织 具体地，本申请提出了 三个目标。 (1)组织培养模型系统将 研究前速激肽原基因在细胞系中的表达 来模拟这个基因的差异表达 在大脑和甲状腺组织中。 (2)序列要求 这种外显子跳跃事件将通过定向 在含有所述突变的基因的区域中的序列的诱变， 任选外显子。 这将包括删除间插序列 和剪接位点区域的诱变。 RNA稳定性的作用 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 指导不同细胞质的产生 前速激肽原mRNA形成。 (3)所需的细胞因子 将通过使用可溶性细胞的生物化学研究来阐明 提取物和模型前mRNA底物构建使用 从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 生物化学研究将提供特异性剪接事件 测试单个剪接位点的内在效率， 剪接位点的背景来源于充分表征的前- mRNA。 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的 过程和组织特异性基因调控。

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.