REGULATED SPLICING OF MESSENGER RNA PRECURSORS

该项目的长期目标是了解该机制 通过它来调节组织特异性剪接事件。 这个 需要阐明顺式作用序列和扩散性序列 细胞因素参与。 为实现这些目标而采取的方法 目标是研究前原速激肽基因的剪接 组织特异性剪接事件的模型。 这是最简单的 可用于这些研究的模型。 该提案的重点是 明显受调控的剪接事件导致跳跃 倒数第二个 3' 外显子优先出现在大脑中，与 甲状腺组织。 具体来说，本申请提出 遵循三个目标。 (1)组织培养模型系统 开发用于研究细胞系中前原速激肽基因表达 模仿该基因差异表达的方式 在脑和甲状腺组织中。 (2)顺序要求 这种外显子跳跃事件将通过定向阐明 含有以下基因的区域中的序列诱变 可选的外显子。 这将包括删除插入序列 和剪接位点区域的诱变。 RNA稳定性的作用 核运输也将在使用这些的研究中得到解决 细胞系以确定剪接事件的程度 指导不同细胞质的产生 前原速激肽 mRNA 形式。 (3)所需的细胞因子 将通过使用可溶性细胞的生化研究来阐明 使用构建的提取物和模型前mRNA底物 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—— 具体剪接事件将由生化研究提供 测试内部各个剪接位点的内在效率 剪接位点的背景源自充分表征的预 mRNA。 对受调控的组织特异性剪接的理解 事件对于全面了解发育至关重要 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.