Interpreting the Structure of the Spliceosome

解释剪接体的结构

• 批准号: 7238022
• 负责人: Melissa S Jurica
• 金额: $ 30.61万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7238022
• 关键词: Active Sites; Address; Affect; Alternative Splicing; Binding; Biochemical; Biochemistry; Catalysis; Chemicals; Chemistry; Cleaved cell; Code; Complex; Cryoelectron Microscopy; Deltastab; Development; Distant; Electron Microscopy; Excision; Exons; Gene Expression; Gene Expression Regulation; Genetic; Goals; Heavy Metals; Heterogeneity; Heterogeneous Nuclear RNA; Image; In Vitro; Introns; Investigation; Isotopes; Label; Ligation; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Melissa; Methods; Modeling; Mutation; Nature; Nuclear; Numbers; Oligonucleotides; Peptides; Preparation; Process; Protein Binding; Proteins; RNA; RNA Sequences; RNA Splicing; Range; Reading Frames; Research Personnel; Resolution; Ribonuclease H; Role; Sampling; Series; Site; Spliceosome Assembly Pathway; Spliceosomes; Structure; Techniques; Transcript; Translations; human disease; mRNA Precursor; macromolecular assembly; numb protein; particle; programs; research study

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.

描述（由申请人提供）：前MRNA处理是真核基因表达的重要步骤。从Messenger RNA的前体（PRE-MRNA）的中间序列（内含子）的本构剪接对于建立正确的翻译读取框是必要的。另外，从同一转录本的替代包含不同的编码序列（外显子）作为基因调节的关键点。影响构成和替代剪接的突变与包括癌症在内的许多人类疾病有关。该提案的目的是获取和解释剪接体的结构信息，这是负责拼接催化的非常大的大分子机器。需要对该重要分子的三维（3D）结构理解，以阐明这种动态复合物如何能够准确地识别沿前MRNA和坐标内含子切除和外显子结扎的非常遥远的剪接位点。因为剪接体是一种动态复合物，由五个结构RNA（U富含U1，U1，U2，U4，U5和U6 SNRNA）和100个蛋白质的顺序组成，因此它给结构研究带来了挑战。冷冻电子显微镜（Cryo-EM）提供了一种可视化这款复杂机器的方法。我们将追求EM标记和生化特征的结合，这些纯化的剪接体的生化表征在剪接化学的两个化学步骤之间捕获，以提供对冷冻EM结构的解释。这将使我们能够在结构上绘制剪接组件，以识别前MRNA底物和活性位点。这些研究将使我们更接近定义剪接位点识别，剪接体组装和剪接催化的机制。