Interpreting the Structure of the Spliceosome

描述（由申请人提供）：前体mRNA加工是真核基因表达的重要步骤。从信使RNA（pre-mRNAs）的前体组成性剪接插入序列（内含子）对于建立用于翻译的正确阅读框架是必需的。此外，来自相同转录物的不同编码序列（外显子）的选择性包含使剪接成为基因调控的关键点。影响组成性剪接和选择性剪接两者的突变与许多人类疾病（包括癌症）相关。这项计划的目标是获得和解释剪接体的结构信息，剪接体是负责剪接催化的非常大的大分子机器。一个三维（3D）结构的理解，这一重要的分子将是必要的，以阐明这种动态复合物是如何能够精确地识别非常遥远的剪接位点沿着前mRNA和协调内含子切除和外显子连接。由于剪接体是由五种结构RNA（富含U的小核U1，U2，U4，U 5和U6 snRNA）和大约100种蛋白质组成的动态复合物，因此它对结构研究提出了挑战。冷冻电子显微镜（cryo-EM）提供了一种可视化这种复杂机器的方法。我们将追求的EM标记和纯化的剪接体之间的剪接化学的两个化学步骤逮捕的生化表征的组合，以提供一个解释的冷冻EM结构。这将使我们能够映射剪接体组件的结构，以确定前mRNA的底物和活性位点。这些研究将使我们更接近于确定剪接位点识别、剪接体组装和剪接催化的机制。

DESCRIPTION (provided by applicant): Pre-mRNA processing is an essential step in eukaryotic gene expression. Constitutive splicing of intervening sequences (introns) from precursors of messenger RNAs (pre-mRNAs) is necessary to establish the correct reading frame for translation. Additionally, alternative inclusion of different coding sequences (exons) from the same transcript places splicing as a pivotal point of gene regulation. Mutations affecting both constitutive and alternative splicing are associated with a number of human diseases, including cancers. The goal of this proposal is to obtain and interpret structural information for the spliceosome, the very large macromolecular machine responsible for splicing catalysis. A three-dimensional (3D) structural understanding of this important molecule will be necessary to elucidate how this dynamic complex is able to precisely recognize very distant splice sites along a pre-mRNA and coordinate intron excision and exon ligation. Because the spliceosome is a dynamic complex composed of five structural RNAs (the U-rich small nuclear U1, U2, U4, U5 and U6 snRNAs) and on the order of 100 proteins, it presents challenges to structural studies. Cryo-electron microscopy (cryo-EM) provides a means to visualize this complicated machine. We will pursue a combination of EM labeling and biochemical characterization of purified splicesomes arrested between the two chemical steps of splicing chemistry to provide an interpretation of the cryo-EM structure. This will allow us to map spliceosome components on the structure to identify the pre-mRNA substrate and active site. These studies will move us closer to defining the mechanisms of splice site identification, spliceosome assembly, and splicing catalysis.