Interpreting the Structure of the Spliceosome

描述(申请人提供)：前信使核糖核酸加工是真核基因表达中必不可少的一步。为了建立正确的翻译阅读框架，必须从信使RNA(前mRNAs)的前体中结构性地剪接插入序列(内含子)。此外，来自同一转录物的不同编码序列(外显子)的替代包含使剪接成为基因调控的关键点。影响结构性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。这项提议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高分子机器。对这一重要分子的三维(3D)结构的了解将是必要的，以阐明这种动态复合体如何能够准确地识别沿着Pre-mRNA非常远的剪接位置，并协调内含子切除和外显子连接。由于剪接体是由5个结构RNA(富含U的小核U1、U2、U4、U5和U6SnRNAs)组成的动态复合体，大约有100个蛋白质，这给结构研究带来了挑战。低温电子显微镜(Cryo-EM)提供了一种可视化这台复杂机器的方法。我们将结合EM标记和生物化学特征对滞留在剪接化学的两个化学步骤之间的纯化剪接体进行研究，以提供对低温EM结构的解释。这将使我们能够将剪接体成分映射到结构上，以确定前mRNA底物和活性部位。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.