Characterization of a minimal "spliceosome"

最小“剪接体”的表征

• 批准号: 7136525
• 负责人: Saba Valadkhan
• 金额: $ 27.04万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7136525
• 关键词: RNA binding protein; RNA splicing; active sites; catalyst; functional /structural genomics; gene expression; genetic manipulation; genetic mapping; genetic regulation; intermolecular interaction; introns; molecular assembly /self assembly; nuclease; nucleobase; nucleotide analog; phosphates; posttranslational modifications; small nuclear RNA; spliceosomes

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to understand, in detail, the role of spliceosomal snRNAs in catalysis of the splicing reaction. In the active spliceosome, U6 and U2 snRNAs form a base-paired complex that helps position the reactants of the first step of splicing for catalysis. Previously published work and preliminary studies show that an in vitro-assembled, base paired U6/U2 complex resembling the one forming in vivo can catalyze a reaction closely related to the first step of splicing. This finding will be pursued under three specific aims. 1) Characterization of the splicing-related catalytic activity of U6 and U2 snRNAs. To conclusively demonstrate that the reaction catalyzed by the in vitro-assembled U6/U2 complex is indeed identical to the first step of splicing, the chemistry of the reaction will be directly analyzed by nuclease digestion of site-specifically labeled products, followed by TLC analysis. In addition, by manipulating the structure of the U6/U2 complex, the basis for the selection of the scissile phosphate will be defined. 2) Determination of the structural organization of the catalytic U6/U2 complex. In vivo data has indicated the importance of the three-dimensional structure of the U6/U2 complex in spliceosomal catalysis. Using a battery of chemical probing reagents and crosslinking assays, the structural architecture of the in vitro- assembled U6/U2 and its interactions with the splicing substrates will be defined. 3) Functional analysis of the sequence elements required for catalysis. Mutational analysis of the snRNAs in the spliceosome has revealed a functionally crucial role for a number of nucleobases and backbone phosphates. The molecular basis of the function of these required elements will be defined using nucleotide analog interference mapping (NAIM). Using a similar approach, the functional significance of abundant post-transcriptional modifications in the U6 and U2 snRNAs will be defined. Almost all human pre-messenger RNAs undergo multiple splicing events, and alternative splicing is not only one of the most important means of regulation of gene expression, it is also largely responsible for generating proteomic diversity in eukaryotes. Disturbances in the pattern of pre-mRNA splicing have been linked to a broad spectrum of human diseases ranging from genetic and neurodegenerative diseases to malignancies.

描述（由申请人提供）：拟议研究的长期目标是详细了解剪接体SNRNA在剪接反应催化中的作用。在主动剪接体中，U6和U2 SNRNA形成了一种基本配合的复合物，该复合物有助于定位剪辑的第一步的反应物。先前发表的工作和初步研究表明，类似于体内形成的一个形成的一个体外组装的，成对的U6/U2复合物可以催化与剪接的第一步密切相关的反应。这一发现将在三个具体目标下进行。 1）表征U6和U2 SNRNA的剪接相关催化活性。最终证明，由体外组装的U6/U2复合物催化的反应确实与剪接的第一步相同，将通过对位点特异性标记的产物的核酸酶消化，然后进行TLC分析，直接分析反应的化学。另外，通过操纵U6/U2复合物的结构，将定义剪刀磷酸盐的选择的基础。 2）确定催化U6/U2复合物的结构组织。体内数据表明U6/U2复合物在剪接体催化中的三维结构的重要性。使用一系列化学探测试剂和交联测定，将定义体外组装的U6/U2的结构结构及其与剪接基板的相互作用。 3）催化所需的序列元件的功能分析。对剪接体中SNRNA的突变分析揭示了许多核碱基和骨链磷酸盐的功能至关重要。这些所需元素功能的分子基础将使用核苷酸类似干扰映射（NAIM）定义。使用类似的方法，将定义丰富的转录后修饰的功能意义。几乎所有人类的预选赛前RNA都会经历多个剪接事件，而替代剪接不仅是调节基因表达的最重要方法之一，而且还主要负责在真核生物中产生蛋白质组学多样性。前MRNA剪接模式的干扰与从遗传和神经退行性疾病到恶性肿瘤的广泛人类疾病有关。