THE STRUCTURE AND COMPOSITION OF THE HUMAN SPLICEOSOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。

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. 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.