Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay
Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay
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DOI：
10.1016/j.yexcr.2011.05.027
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发表时间：
2011-09-10
影响因子：
3.7
通讯作者：
Yang, Wei-Hong
中科院分区：
文献类型：
--
作者：
Bloch, Donald B.;Nobre, Rita A.;Yang, Wei-Hong
mRNA加工体（P体）的组分调节mRNA储存、运输、翻译和降解的关键步骤。P体的核心是脱帽复合物，其从脱腺苷酸化的mRNA中除去5'帽并介导mRNA降解中的不可逆步骤。先前已经描述了酿酒酵母、拟南芥和黑腹果蝇中P体的组装。对哺乳动物P体的组装知之甚少。为了研究哺乳动物P-小体组分之间的相互作用，我们开发了一种基于荧光的双杂交检测系统。该测定取决于一个P体组分与外源性核定位序列（NLS）融合的能力，以招募其他P体组分到细胞核。该测定用于研究P体组分Ge-1、DCP 2、DCP 1、EDC 3、RAP 55和RCK之间的相互作用。本研究的结果表明，修改后的双杂交试验可以用来确定蛋白质的相互作用，发生在一个大分子复合物。该测定也可用于有效地检测蛋白质相互作用结构域。结果提供了重要的见解哺乳动物P-体组装和展示的相似性，和关键的差异，在哺乳动物细胞中的P-体组装与其他物种相比。(C)2011 Elsevier Inc. All rights reserved.
Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. (C) 2011 Elsevier Inc. All rights reserved.