Interpreting the Structure of the Spliceosome

描述（由申请人提供）：Pre-mRNA加工是真核生物基因表达的重要步骤。来自信使rna前体（pre- mrna）的干预序列（内含子）的组成剪接对于建立正确的翻译阅读框架是必要的。此外，来自同一转录本的不同编码序列（外显子）的替代包含使剪接成为基因调控的关键点。影响构象剪接和选择性剪接的突变与包括癌症在内的许多人类疾病有关。本提案的目标是获取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。对这一重要分子的三维（3D）结构理解将是必要的，以阐明这种动态复合物如何能够精确地识别非常远的剪切位点，并协调内含子切除和外显子连接。由于剪接体是由5种结构rna（富含u的核小rna U1、U2、U4、U5和U6 snRNAs）组成的动态复合体，约为70-100个蛋白，因此对其结构研究存在很大障碍。低温电子显微镜（cryo-EM）提供了一种可视化这种复杂机器的手段。我们将对剪接化学的两个化学步骤之间捕获的纯化剪接体进行EM标记和生化表征的结合，以提供冷冻电镜结构的解释。这将使我们能够在结构上绘制剪接体成分，以识别前mrna底物和活性位点。这些研究将使我们更接近于定义剪接位点识别、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 attain 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 70-100 proteins, it presents large hurdles 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 spliceosomes 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.