Comprehensive proteomic analysis of the human spliceosome
Comprehensive proteomic analysis of the human spliceosome
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DOI：
10.1038/nature01031
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发表时间：
2002-09-12
期刊：
影响因子：
64.8
通讯作者：
Reed, R
中科院分区：
文献类型：
--
作者：
Zhou, ZL;Licklider, LJ;Reed, R
从前信使RNA中精确地切除内含子是由剪接体执行的，剪接体是一个包含五个小核RNA和大量蛋白质的大分子机器。通过对单个纯化的小核核糖核蛋白和盐稳定性剪接体‘核心’颗粒的分析，已经对剪接体的蛋白质成分有了很多了解。然而，构成完整功能剪接体的一整套蛋白质尚未确定。在这里，我们使用麦芽糖结合蛋白亲和层析来分离高纯度和功能性的剪接体。利用纳米级微毛细管液相色谱串联质谱仪，我们鉴定了145种截然不同的剪接体蛋白，使剪接体成为迄今表征的最复杂的细胞机。我们的剪接体包括所有已知的剪接因子和58个新发现的组分。剪接体包含至少30种蛋白质，这些蛋白质在剪接以外的基因表达步骤中具有已知或推测的作用。这种复杂性可能不仅是剪接多内含子后生动物前信使RNA所必需的，也是调节剪接和基因表达中其他步骤之间的广泛耦合所必需的。
The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify similar to145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.