Makino Shiho, Mishima Yuichiro, Inoue Kunio, Inada Toshifumi
From the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.
the Institute of Molecular and Cellular Biosciences and the Department of Medical Genome Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan, and
J Biol Chem. 2015 Mar 27;290(13):8331-47. doi: 10.1074/jbc.M114.615088. Epub 2015 Feb 5.
The CCR4-NOT complex, the major deadenylase in eukaryotes, plays crucial roles in gene expression at the levels of transcription, mRNA decay, and protein degradation. GW182/TNRC6 proteins, which are core components of the microRNA-induced silencing complex in animals, stimulate deadenylation and repress translation via recruitment of the CCR4-NOT complex. Here we report a heterologous experimental system that recapitulates the recruitment of CCR4-NOT complex by TNRC6 in S. cerevisiae. Using this system, we characterize conserved functions of the CCR4-NOT complex. The complex stimulates degradation of mRNA from the 5' end by Xrn1, in a manner independent of both translation and deadenylation. This degradation pathway is probably conserved in miRNA-mediated gene silencing in zebrafish. Furthermore, the mRNA fate modulators Dhh1 and Pat1 redundantly stimulate mRNA decay, but both factors are required for poly(A) tail-independent translation repression by tethered TNRC6A. Our tethering-based reconstitution system reveals that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting microRNA-induced silencing complex to the decapping machinery as well as the translation apparatus.
CCR4-NOT复合物是真核生物中的主要去腺苷酸化酶，在转录、mRNA降解和蛋白质降解水平的基因表达中发挥关键作用。GW182/TNRC6蛋白是动物中微小RNA诱导沉默复合物的核心成分，通过招募CCR4-NOT复合物来刺激去腺苷酸化并抑制翻译。在此，我们报告了一种异源实验系统，该系统概括了酿酒酵母中TNRC6对CCR4-NOT复合物的招募。利用该系统，我们表征了CCR4-NOT复合物的保守功能。该复合物通过Xrn1从5'端刺激mRNA的降解，其方式独立于翻译和去腺苷酸化。这种降解途径可能在斑马鱼的微小RNA介导的基因沉默中是保守的。此外，mRNA命运调节因子Dhh1和Pat1冗余地刺激mRNA降解，但这两个因子都是通过拴系的TNRC6A进行不依赖于聚腺苷酸尾的翻译抑制所必需的。我们基于拴系的重组系统表明，Not1/CNOT1的保守结构为TNRC6提供了一个结合表面，从而将微小RNA诱导沉默复合物与脱帽机制以及翻译装置连接起来。
