Understanding the structure and function of splicing factor Prp8

描述（由申请人提供）：前体mRNA剪接对于所有真核生物中的基因表达是必不可少的，剪接错误会导致遗传疾病和许多其他疾病。例如，剪接因子Prp 8的突变会导致一种严重的人类遗传性疾病视网膜色素变性。对前体mRNA剪接的分子机制的透彻理解有可能为人类疾病治疗提供有用的方法。内含子的剪接是通过剪接体催化的两个酯交换反应进行的，剪接体是由5个snRNA和100多个蛋白质因子组成的大型RNA/蛋白质复合物。许多证据表明Prp 8是一种关键的剪接体蛋白，它在催化核心与RNA密切相互作用，可能有助于催化核心的形成和稳定。Prp 8是已知的最大和最保守的核蛋白之一，但它与任何其他已知蛋白没有明显的序列同源性。进一步的结构和生物化学分析将为Prp 8在剪接中的功能提供有价值的见解。然而，这些研究受到难以获得大量全长Prp 8的阻碍。鉴定、表达和纯化Prp 8的结构域将为表征Prp 8提供有价值的替代方法。该建议使用独特的高通量方法来鉴定可以在大肠杆菌中以可溶形式表达的Prp 8结构域。coli中的结构域并确定其结构。这些结构域的结构以及与其他已知结构的比较可以提供关于Prp 8在剪接中的功能的重要信息，指导未来的突变/遗传实验。这些可溶性结构域也是表征Prp 8的生物化学性质的宝贵资源，例如其与RNA、其他蛋白质伴侣以及不同Prp 8结构域之间的相互作用。一旦完成单个结构域的结构和生化表征，就可以对组成多个结构域的Prp 8区域进行类似的表征。这种方法是产生Prp 8的完整图像的关键一步，否则无法获得，显着推进我们对前体mRNA剪接的分子机制的理解。

DESCRIPTION (provided by applicant): 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.