The DDX6-4E-T interaction mediates translational repression and P-body assembly.
The DDX6-4E-T interaction mediates translational repression and P-body assembly.
复制标题
DOI：
10.1093/nar/gkw565
复制
发表时间：
2016-07-27
影响因子：
14.9
通讯作者：
Standart N
中科院分区：
文献类型：
--
作者：
Kamenska A;Simpson C;Vindry C;Broomhead H;Bénard M;Ernoult-Lange M;Lee BP;Harries LW;Weil D;Standart N
4 E-转运蛋白通过其与eIF 4G共有的共有序列YXXXXLΦ结合eIF 4 E，并且是一种发现在P-（加工）体中富集的核质穿梭蛋白。4 E-T通过降低可用的eIF 4 E水平来抑制一般蛋白质合成。最近，我们发现，4 E-T结合mRNA，但抑制其翻译的eIF 4 E-独立的方式，并有助于沉默的mRNA靶向的miRNA。在这里，我们进一步解决的翻译抑制4 E-T的机制，首先确定和描绘其主要合作伙伴的相互作用位点的质谱和蛋白质印迹，包括DDX 6，UNR，unrip，PAT 1B，LSM 14 A和hocT 4。此外，我们记录了4 E-T伙伴之间的新结合，包括E-hocT 4和unrip-LSM 14 A，这表明4 E-T使RNA结合蛋白相互作用的复杂网络成核。在功能测定中，我们证明了结合UNR和DDX 6的两个短保守基序的联合缺失缓解了4 E-T结合mRNA的抑制，部分依赖于4 E-T-DDX 6-hocT 1轴。我们还发现DDX 6 - 4 E-T相互作用介导了miRNA依赖的翻译抑制和从头P体组装，这意味着翻译抑制和新P体的形成是耦合的过程。总之，这些发现大大扩展了我们对4 E-T在基因调控中的作用的理解，这在发育和神经发生中很重要。
4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.