Interpreting the Structure of the Spliceosome

描述（由申请人提供）：前体mRNA剪接是真核基因表达的重要步骤，是调控的关键点。剪接从信使RNA的前体（前mRNA）中去除插入内含子序列以建立mRNA翻译的正确阅读框架。此外，通过选择性剪接的外显子编码序列的差异包含极大地扩展了基因的信息潜力。影响剪接的突变与许多人类疾病有关，包括癌症。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。这些信息对于理解剪接体如何能够精确地识别沿着前mRNA的非常远的剪接位点并协调内含子切除和外显子连接是必要的。由于剪接体是由5种结构RNA（富含U的小核U1、U2、U4、U5和U6 snRNA）和100多种蛋白质组成的动态复合体，因此它对结构研究提出了特殊的挑战。我们将结合联合收割机先进的质谱方法和电子显微镜研究，以检查剪接体在不同阶段相对于剪接化学逮捕。通过比较不同的构象，我们将确定许多剪接体组件是如何排列的，并创建剪接体结构的模型。这些研究对于理解剪接位点识别、剪接体组装和剪接催化的机制至关重要。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is an essential step in eukaryotic gene expression and serves as a key point of regulation. Splicing removes intervening intron sequences from precursors of messenger RNAs (pre- mRNA) to establish the correct reading frame for mRNA translation. Additionally, differential inclusion of exon coding sequences by alternative splicing vastly expands information potential of genes. Mutations that affect 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. This information is necessary to understand how the spliceosome 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 over of 100 proteins, it presents special challenges to structural studies. We will combine sophisticated mass spectrometry approaches and electron microscopy studies to examine spliceosomes arrested at different stages relative to splicing chemistry. By comparing the different conformations, we will determine how the many spliceosome components are arranged and create models of spliceosome structure. These studies are critical to understanding mechanisms of splice site identification, spliceosome assembly, and splicing catalysis.