Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes
Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes
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DOI：
10.1128/mcb.01415-08
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发表时间：
2009-01-01
影响因子：
5.3
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Herold, Nadine;Will, Cindy L.;Luehrmann, Reinhard
剪接体复合物的综合蛋白质组学分析目前仅限于人类，因此，目前还不清楚剪接体的高度复杂的组成和组成动力学在多细胞动物中的保守程度。在这里，我们亲和纯化果蝇剪接体B和C复合物形成的KC细胞核提取物。质谱分析显示，它们的组成与人类B和C复合物的组成高度相似。尽管如此，一些果蝇特有的蛋白质被鉴定出来，这表明可能有新的因素对果蝇的剪接有特异性的贡献。在B到C的过渡过程中的蛋白质招募和释放事件也非常相似，在这两种生物体。果蝇B复合物的电子显微镜显示与人类B复合物的高度结构相似性，表明高阶相互作用也在很大程度上是保守的。果蝇剪接体形成的一个短与长内含子的比较显示，只有很小的差异，蛋白质组成，但尽管如此，在电子显微镜下，明确的结构差异。最后，亲和纯化的果蝇mRNP的表征表明，外显子连接复合体蛋白在C复合体形成过程中以剪接依赖的方式被招募。这些研究提供了深入了解进化保守的后生动物剪接体的组成和结构，以及催化活化过程中的组成动态。
Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.