Molecular Architecture of the Human Prp19/CDC5L Complex
Molecular Architecture of the Human Prp19/CDC5L Complex
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DOI：
10.1128/mcb.01505-09
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发表时间：
2010-05-01
影响因子：
5.3
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Grote, Michael;Wolf, Elmar;Luehrmann, Reinhard
含有Prp 19的蛋白质复合物在剪接体的催化活化过程中发挥核心作用，并且Prp 19及其相关蛋白是剪接体的催化核心RNP的主要组分。为了了解更多关于人Prp 19（hPrp 19）/CDC 5L复合物（由hPrp 19、CDC 5L、PRL 1、AD 002、SPF 27、CTNNBL 1和HSP 73组成）的空间组织的信息，我们从稳定表达FLAG标记的AD 002或SPF 27的HeLa细胞中纯化了天然hPrp 19/CDC 5L复合物。化学计量分析表明，像酿酒酵母NTC（十九复合物），人Prp 19/CDC 5L复合物含有四个拷贝的hPrp 19。盐处理鉴定了由CDC 5L、hPrp 19、PRL 1和SPF 27组成的稳定核心。蛋白质-蛋白质相互作用研究表明，SPF 27直接与hPrp 19/CDC 5L复合物核心的每种组分相互作用，并阐明了hPrp 19/CDC 5L复合物组分之间的几种额外的、先前未知的相互作用。有限的蛋白水解的hPrp 19/CDC 5L复合物揭示了蛋白酶抗性复合物组成的SPF 27，C末端的CDC 5L，和N末端的PRL 1和hPrp 19。在电子显微镜下，纯化的hPrp 19/CDC 5L复合物表现出细长的，不对称的形状，最大尺寸接近20 nm。我们的研究结果不仅阐明了hPrp 19/CDC 5L复合物的分子组织，而且还提供了对催化活性剪接体核心的潜在蛋白质-蛋白质相互作用的见解。
Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of similar to 20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.