Understanding the structure and function of splicing factor Prp8

描述（由申请人提供）：Pre-mRNA剪接对于所有真核生物的基因表达至关重要，剪接错误会导致遗传疾病和许多其他疾病。例如，剪接因子Prp8的突变会导致一种严重的人类遗传性疾病色素性视网膜炎。对前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次酯交换反应进行的，剪接体是由5个snrna和100多个蛋白质因子组成的大型RNA/蛋白质复合体。许多证据表明，Prp8是一个关键的剪接体蛋白，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是目前已知的最大、最保守的核蛋白之一，但它与其他已知蛋白没有明显的序列同源性。进一步的结构和生化分析将为Prp8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Prp8的阻碍。鉴定、表达和纯化Prp8结构域将为表征Prp8提供一种有价值的替代方法。本研究采用一种独特的高通量方法来鉴定可在大肠杆菌中以可溶性形式表达的Prp8结构域，并确定这些结构域的结构。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Prp8在剪接中的功能的重要信息，指导未来的突变/遗传实验。这些可溶结构域也是表征Prp8生化特性的宝贵资源，例如它与RNA、其他蛋白质伙伴以及不同Prp8结构域之间的相互作用。一旦完成了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Prp8区域进行类似的表征。该方法是获得Prp8完整图谱的关键一步，这是其他方法无法获得的，它极大地促进了我们对pre-mRNA剪接分子机制的理解。

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.