THE STRUCTURE AND COMPOSITION OF THE HUMAN SPLICEOSOME

人类剪接体的结构和组成

• 批准号: 7724212
• 负责人: Melissa S Jurica
• 金额: $ 0.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724212
• 关键词: Binding; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Funding; Genes; Grant; Hela Cells; Heterogeneous Nuclear RNA; Human; In Vitro; Institution; Introns; Isotopes; Mass Spectrum Analysis; Nuclear Extract; Peptides; Protein Binding; Proteins; Protocols documentation; RNA Splicing; Research; Research Personnel; Resolution; Resources; Sampling; Series; Source; Spliceosomes; Structure; Surface; Transcript; United States National Institutes of Health; instrumentation; mRNA Precursor; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The spliceosome is the cellular machine responsible for removing the introns that interrupt nearly all human gene transcripts. In a highly dynamic series of molecular interactions, the spliceosome is assembled on each intron from on the order of 150 proteins, as well as 5 structural RNAs. The Jurica group has developed a protocol isolate to spliceosomes assembled in vitro on a synthetic splicing substrate from HeLa cell nuclear extract. LC-MS/MS analysis of these complexes identified a potential "parts list" of over 200 proteins, although the stoichiometric presence of many of these proteins remains to be verified. Control experiments indicate that a subset of the proteins bind the pre-mRNA even in the absence of splicing. In order to further define the composition of the core spliceosome complex Jurica will use mass spectrometry to identify proteins associated with the spliceosome when flanking pre-mRNA is released. They will also compare the composition of C complex assembled on other pre-mRNA splicing substrates and employ isotope tagging to allow more quantitative comparison of these different samples. Additionally, they will identify proteins that are exposed on the surface of the spliceosome by chemically modifying purified spliceosome complexes under both native and denaturing conditions. Using mass spectrometry to analyze peptides derived from these samples, they can conclude that peptides modified in both samples are surface exposed, while those modified only in the denatured complexes are buried within the complex. The limiting amount of material and high complexity of their samples will require mass spectrometric instrumentation with very high resolution and very high sensitivity.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 剪接体是负责去除几乎所有人类基因转录本的内含子的细胞机。 在一系列高度动态的分子相互作用中，剪接体在每个内含子上的150蛋白和5个结构RNA都组装在每个内含子上。 Jurica组已开发出一种在HeLa细胞核提取物的合成剪接底物上在体外组装的分离体的方案。 这些复合物的LC-MS/MS分析确定了200多个蛋白质的潜在“零件清单”，尽管这些蛋白质中许多蛋白的化学计量存在仍然有待验证。 对照实验表明，即使在没有剪接的情况下，蛋白质的子集也会结合前MRNA。 为了进一步定义核心剪接体复合物的组成，当释放前MRNA时，将使用质谱法来鉴定与剪接体相关的蛋白质。 他们还将比较在其他前MRNA剪接底物上组装的C复合物的组成，并采用同位素标记，以便对这些不同样品进行更定量的比较。 此外，他们将通过化学修饰纯化的剪接体复合物在天然和变性条件下化学修饰纯化的剪接体复合物，从而鉴定出在剪接体表面暴露的蛋白质。 使用质谱法分析从这些样品中得出的肽，他们可以得出结论，在两个样品中修饰的肽都表面暴露，而仅在变性复合物中修饰的肽则埋在复合物中。 材料的限制量和样品的高复杂性需要具有很高的分辨率和非常高灵敏度的质谱仪器。