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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 剪接体是负责去除内含子的细胞机器，内含子几乎中断了所有人类基因转录。 在一系列高度动态的分子相互作用中，剪接体从大约150种蛋白质以及5种结构RNA组装在每个内含子上。 Jurica小组已经开发出一种分离方案，用于在HeLa细胞核提取物的合成剪接底物上体外组装剪接体。 这些复合物的LC-MS/MS分析确定了超过200种蛋白质的潜在“部分列表”，尽管这些蛋白质中的许多的化学计量存在仍有待验证。 对照实验表明，即使在没有剪接的情况下，蛋白质的一个子集也与前mRNA结合。 为了进一步确定核心剪接体复合物的组成，Jurica将使用质谱法来鉴定当侧翼前mRNA释放时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，并采用同位素标记来对这些不同的样品进行更定量的比较。 此外，他们将通过在天然和变性条件下化学修饰纯化的剪接体复合物来鉴定暴露在剪接体表面上的蛋白质。 使用质谱分析来自这些样品的肽，他们可以得出结论，在两个样品中修饰的肽是表面暴露的，而那些仅在变性复合物中修饰的肽被埋在复合物中。 有限的材料量和样品的高度复杂性将需要具有非常高的分辨率和非常高的灵敏度的质谱仪器。