THE STRUCTURE AND COMPOSITION OF THE HUMAN SPLICEOSOME

人类剪接体的结构和组成

• 批准号: 8363769
• 负责人: Melissa S Jurica
• 金额: $ 0.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363769
• 关键词: Binding; Complex; Funding; Genes; Grant; Hela Cells; Human; In Vitro; Introns; Mass Spectrum Analysis; National Center for Research Resources; Nuclear Extract; Peptides; Principal Investigator; Protein Binding; Proteins; Protocols documentation; RNA Splicing; Research; Research Infrastructure; Resolution; Resources; Sampling; Series; Source; Spliceosomes; Structure; Surface; Transcript; United States National Institutes of Health; cost; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. 1) 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. 2) They will also compare the composition of spliceosomes arrested at other points of splicing. 3) In addition, 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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 剪接体是负责移除干扰几乎所有人类基因转录的内含子的细胞机器。在一系列高度动态的分子相互作用中，剪接体组装在来自150个蛋白质和5个结构RNA的每个内含子上。Jurica团队已经开发出一种方案，可以将体外组装在HeLa细胞核提取液合成剪接底物上的小体剪接起来。对这些复合体的LC-MS/MS分析确定了一个潜在的200多种蛋白质的“部分清单”，尽管其中许多蛋白质的化学计量存在仍有待证实。对照实验表明，即使在没有剪接的情况下，也有一部分蛋白质与前-mRNA结合。1)为了进一步确定核心剪接体复合体的组成，Jurica将在侧翼释放Pre-mRNA时使用质谱仪鉴定与剪接体相关的蛋白质。2)他们还将比较在其他剪接点停滞的剪接体的组成。3)此外，他们将通过在天然和变性条件下对纯化的剪接体复合体进行化学修饰来鉴定暴露在剪接体表面的蛋白质。用质谱仪分析这些样品中的多肽，他们可以得出结论，在两个样品中修饰的多肽都是表面暴露的，而那些只在变性复合体中修饰的多肽隐藏在复合体中。材料的数量有限和样品的高度复杂性将需要非常高的分辨率和非常高的灵敏度的质谱仪。