Characterization of purified human B act spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis
Characterization of purified human B act spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis
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DOI：
10.1261/rna.2456210
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发表时间：
2010-12-01
期刊：
影响因子：
4.5
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Bessonov, Sergey;Anokhina, Maria;Luehrmann, Reinhard
为了更好地了解人类剪接体在其激活过程中的组成和结构动力学，我们开始分离在预催化B之后形成的剪接体复合物，但在催化活性C复合物之前形成。通过缩短PM5 pre- mRNA的聚嘧啶束（缺乏3‘剪接位点和3’外显子），我们阻止了剪接体在激活阶段的组装。随后，我们在先前用于分离B和C复合物的相同条件下亲和纯化了人B act复合物，并通过质谱分析了它们的蛋白质组成。对这些复合物的蛋白质组成进行比较，可以很好地解剖B到B行为和B行为到C转变过程中的组成变化，与酿酒酵母B行为复合物的比较表明，剪接体在激活过程中的组成动力学在低等和高等真核生物之间很大程度上是保守的。人类SF3b155和CDC5L分别在B - to - B行为和B - act - to - C转变过程中被特异性磷酸化，表明这些修饰在剪接的这些阶段起作用。通过电子显微镜测定了人B行为复合物的二维结构，并与B复合物进行了比较，发现人剪接体在其激活过程中形态发生了显著变化。人类和酿酒酵母B行为复合体的整体结构是相似的，这表明剪接体组分之间的许多高阶相互作用及其动力学在很大程度上也是保守的。
To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre- mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B act complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B act and B act to C transitions, and comparisons with the Saccharomyces cerevisiae B act complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B act and B act to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B act complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B act complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.