Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions
Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions
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DOI：
10.1128/mcb.00582-06
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发表时间：
2006-07-01
影响因子：
5.3
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Deckert, Jochen;Hartmuth, Maus;Luehrmann, Reinhard
剪接体 B 复合物是经历催化激活的底物，导致前 mRNA 剪接的催化。先前对该复合物的表征是在肝素存在下进行的，肝素会解离不稳定的相关成分。为了获得 B 复合体蛋白质组的更全面的清单，我们使用两种独立的方法在低严格条件下分离了该复合体。当在缺乏 snRNP 的核提取物中孵育时，MS2 亲和选择的 B 复合物支持剪接。质谱法在两种独立纯化的 B 复合物制剂中鉴定出了超过 110 种蛋白质，其中包括类似于之前在剪接体 A 复合物中未发现的 50 种非 snRNP 蛋白质。出乎意料的是，检测到异聚 hPrp19/CDC5 复合物和 10 个额外的 hPrp19/CDC5 相关蛋白，表明它们在剪接体激活之前被招募。电子显微镜研究表明，MS2 亲和选择的 B 复合物呈现最大尺寸为 420 A 的菱形形状，并且在结构上比用肝素处理的 B 复合物更均匀。这些数据为剪接体在催化激活之前的组成和结构提供了新的见解，并表明了在此阶段招募的蛋白质激活中的潜在作用。此外，这里分离的剪接体复合物非常适合与纯化的蛋白质进行互补研究，以剖析剪接体激活和剪接催化的因子要求。
The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including similar to 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.