P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes.
P-body assembly requires DDX6 repression complexes rather than decay or Ataxin2/2L complexes.
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DOI：
10.1091/mbc.e15-03-0136
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发表时间：
2015-07-15
影响因子：
3.3
通讯作者：
Weil D
中科院分区：
文献类型：
--
作者：
Ayache J;Bénard M;Ernoult-Lange M;Minshall N;Standart N;Kress M;Weil D
DDX 6是一种丰富的DEAD盒解旋酶，与参与mRNA降解和抑制的各种复合物相关。它在人类细胞中的相互作用组被分析，以确定其最突出的合作伙伴。其中，三种蛋白质是在所有测试条件下P体组装所必需的：DDX 6、4 E-T和LSM 14 A。P体是参与转录后调节的细胞质核糖核蛋白颗粒。DDX 6是它们在人类细胞中组装的关键成分。已知这种DEAD盒RNA解旋酶与各种复合物相关，包括去帽复合物、CPEB抑制复合物、RISC和CCR 4/NOT复合物。为了了解P体组装所需的DDX 6复合物，我们使用串联亲和纯化方法结合质谱分析了DDX 6相互作用组。三个复合物是突出的：脱帽复合物，CPEB样复合物，和Ataxin 2/Ataxin 2L复合物。还发现了外显子连接复合物，表明DDX 6与新输出的mRNA结合。最后，一些DDX 6与多核糖体相关，如先前在酵母中报道的。尽管其在P体中高度富集，但大多数DDX 6定位于P体之外。在这三种复合物中，只有脱帽和CPEB样复合物被招募到P-体中。在各种条件下的P-体组装的调查使我们能够区分所需的蛋白质，从那些被抑制或只参与特定条件。在所有测试条件下需要三种蛋白质：DDX 6、4 E-T和LSM 14 A。这些结果揭示了P-体组装的多种途径，但都有三个关键因素将P-体组装与阻遏联系起来。
DDX6 is an abundant DEAD-box helicase associated with various complexes involved in mRNA decay and repression. Its interactome in human cells was analyzed to identify its most prominent partners. Among them, three proteins were essential for P-body assembly in all tested conditions: DDX6, 4E-T, and LSM14A. P-bodies are cytoplasmic ribonucleoprotein granules involved in posttranscriptional regulation. DDX6 is a key component of their assembly in human cells. This DEAD-box RNA helicase is known to be associated with various complexes, including the decapping complex, the CPEB repression complex, RISC, and the CCR4/NOT complex. To understand which DDX6 complexes are required for P-body assembly, we analyzed the DDX6 interactome using the tandem-affinity purification methodology coupled to mass spectrometry. Three complexes were prominent: the decapping complex, a CPEB-like complex, and an Ataxin2/Ataxin2L complex. The exon junction complex was also found, suggesting DDX6 binding to newly exported mRNAs. Finally, some DDX6 was associated with polysomes, as previously reported in yeast. Despite its high enrichment in P-bodies, most DDX6 is localized out of P-bodies. Of the three complexes, only the decapping and CPEB-like complexes were recruited into P-bodies. Investigation of P-body assembly in various conditions allowed us to distinguish required proteins from those that are dispensable or participate only in specific conditions. Three proteins were required in all tested conditions: DDX6, 4E-T, and LSM14A. These results reveal the variety of pathways of P-body assembly, which all nevertheless share three key factors connecting P-body assembly to repression.