Understanding the structure and function of splicing factor Prp8

前体mRNA剪接对于所有真核生物的基因表达和剪接中的错误都是必不可少的 导致遗传紊乱和许多其他疾病。剪接因子Prp 8的突变， 例如，导致严重的人类遗传疾病视网膜色素变性。的透彻 对前体mRNA剪接的分子机制的理解有可能提供 用于人类疾病治疗的有用方法。内含子的剪接是通过两个 由剪接体（一种大型RNA/蛋白质复合物）催化的酯交换反应 由5种snRNA和100多种蛋白质因子组成。许多证据表明Prp 8是 一种关键的剪接体蛋白，在催化核心与RNA密切相互作用， 有助于催化核的形成和稳定。Prp 8是世界上最大、 已知保守的核蛋白，但它与任何已知的核蛋白都没有明显的序列同源性。 其他已知蛋白质进一步的结构和生化分析将提供有价值的 Prp 8在剪接中的功能。然而，这些研究受到以下困难的阻碍： 获得大量全长Prp 8。鉴定、表达和纯化 Prp 8将为表征Prp 8提供有价值的替代方法。该提案使用一个 一种独特的高通量方法来鉴定Prp 8的结构域， E. coli中的结构域并确定其结构。这些结构域的结构和 与其他已知结构的比较可以提供关于 Prp 8剪接，指导未来的突变/遗传实验。这些可溶性结构域是 也是描述Prp 8生物化学特性的宝贵资源，例如其相互作用 与RNA，其他蛋白质伴侣，以及不同的Prp 8结构域之间。一旦结构和 完成了各个域的生化表征，类似的表征 可以进行Prp 8的区域组成多个结构域。这种方法是关键的一步 为了产生Prp 8的完整图片，否则无法获得，显著 推进我们对前体mRNA剪接的分子机制的理解。前体mRNA剪接对于所有真核生物的基因表达和剪接中的错误都是必不可少的 导致遗传紊乱和许多其他疾病。剪接因子Prp 8的突变， 例如，导致严重的人类遗传疾病视网膜色素变性。的透彻 对前体mRNA剪接的分子机制的理解有可能提供 用于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.