Department of Biochemistry, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany.
Genes Dev. 2019 Feb 1;33(3-4):236-252. doi: 10.1101/gad.320952.118. Epub 2019 Jan 28.
The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or complex. Here we show that the C-terminal regions of human and NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions.
多亚基 CCR4-NOT mRNA 脱腺苷酶复合物在基因表达的转录后调控中发挥重要作用。NOT4 E3 泛素连接酶是酵母中 CCR4-NOT 复合物的稳定组成部分，但不能与人和复合物共纯化。在这里，我们表明人和 NOT4 的 C 末端区域包含一个保守的序列基序，该基序可直接结合 CCR4-NOT 复合物的 CAF40 亚基（CAF40 结合基序 [CBM]）。此外，CBM 侧翼的非保守序列也与复合物的其他亚基接触。CBM-CAF40 复合物的晶体结构揭示了 NOT4 和 Roquin 或 Marble 袋 mRNA 调节蛋白之间的互斥结合表面。此外，CAF40 耗尽或结构导向的突变以破坏 NOT4-CAF40 相互作用会损害 NOT4 在细胞中引发连接的报告 mRNA 衰变的能力。结合其他序列分析，我们的结果揭示了真核生物 NOT4 与 CCR4-NOT 复合物的关联的分子基础，并表明它与酵母有很大的不同。它们将 NOT4 泛素连接酶标记为 CCR4-NOT 脱腺苷酶的古老但非组成性辅因子，具有潜在的募集和/或效应功能。
