Understanding the structure and function of splicing factor Prp8

前信使核糖核酸剪接是所有真核生物中基因表达和剪接错误所必需的 导致遗传性疾病和许多其他疾病。剪接因子Prp8的突变 例如，导致严重形式的人类遗传性疾病视网膜色素变性。一次彻底的 对前mRNA剪接的分子机制的理解有可能为 人类疾病治疗的有用方法。内含子的剪接是通过两个 剪接体--一种大的RNA/蛋白质复合体--催化的酯交换反应 由5个单链RNA和100多个蛋白质因子组成。许多证据表明Prp8是 一种关键的剪接体蛋白，它与催化核心的RNA密切相互作用，可能 有助于催化核心的形成和稳定。Prp8是最大和最多的 已知的保守的核蛋白，但它与任何 其他已知的蛋白质。进一步的结构和生化分析将提供有价值的 洞察Prp8的S在剪接中的作用。然而，这些研究受到以下方面的困难的阻碍 获得大量全长Prp8。鉴定、表达和纯化结构域 Prp8将为表征Prp8提供一种有价值的替代方法。此提案使用一个 用独特的高通量方法确定Prp8的可溶性表达结构域 在大肠杆菌中形成并确定这些结构域的结构。这些域的结构和 与其他已知结构的比较可以提供有关该蛋白功能的重要信息 Prp8参与剪接，指导未来的突变/遗传实验。这些可溶域是 也是表征Prp8‘S生化特性的宝贵资源，如它的相互作用 与RNA、其他蛋白质伙伴，以及不同的Prp8结构域之间。曾经的结构和 完成了单个结构域的生化表征，类似的表征 可以执行组成多个结构域的Prp8区域。这种方法是关键的一步。 对于生成Prp8的完整图景，以其他方式无法获得 促进我们对Pre-mRNA剪接的分子机制的理解。前信使核糖核酸剪接是所有真核生物中基因表达和剪接错误所必需的 导致遗传性疾病和许多其他疾病。剪接因子Prp8的突变 例如，导致严重形式的人类遗传性疾病视网膜色素变性。一次彻底的 对前mRNA剪接的分子机制的理解有可能为 人类疾病治疗的有用方法。

Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. Mutations in splicing factor Prp8, for example, cause a severe form of human genetic disorder Retinitis Pigmentosa. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. Splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNAs and over 100 protein factors. Many lines of evidence point to Prp8 as a key spliceosomal protein that interacts intimately with RNA at the catalytic core, potentially helping the formation and stabilization of the catalytic core. Prp8 is one of the largest and most conserved nuclear proteins known, but it does not have obvious sequence homology with any other known protein. Further structural and biochemical analyses would provide valuable insight into Prp8's function in splicing. However, these studies are hindered by difficulties in obtaining large quantities of full-length Prp8. Identifying, expressing, and purifying domains of Prp8 will provide a valuable alternative approach for characterizing Prp8. This proposal uses a unique high throughput approach to identify domains of Prp8 that can be expressed in soluble forms in E. coli and determine structures of these domains. Structures of these domains and comparison with other known structures can provide important information on the function of Prp8 in splicing, directing future mutational/genetic experiments. These soluble domains are also valuable resources for characterizing Prp8's biochemical properties, such as its interaction with RNA, other protein partners, and among different Prp8 domains. Once structural and biochemical characterizations of individual domains are completed, similar characterizations of regions of Prp8 composing multiple domains can be performed. This approach is a critical step toward generating a complete picture of Prp8 that cannot be obtained otherwise, significantly advancing our understanding of the molecular mechanisms of pre-mRNA splicing. Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. Mutations in splicing factor Prp8, for example, cause a severe form of human genetic disorder Retinitis Pigmentosa. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy.