Department of Biochemistry, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076, Tübingen, Germany.
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany.
Nat Commun. 2019 Jul 18;10(1):3173. doi: 10.1038/s41467-019-11094-z.
CCR4-NOT is a conserved multiprotein complex which regulates eukaryotic gene expression principally via shortening of poly(A) tails of messenger RNA or deadenylation. Here, we reconstitute a complete, recombinant human CCR4-NOT complex. Our reconstitution strategy permits strict compositional control to test mechanistic hypotheses with purified component variants. CCR4-NOT is more active and selective for poly(A) than the isolated exonucleases, CCR4a and CAF1, which have distinct deadenylation profiles in vitro. The exonucleases require at least two out of three conserved non-enzymatic modules (CAF40, NOT10:NOT11 or NOT) for full activity in CCR4-NOT. CAF40 and the NOT10:NOT11 module both bind RNA directly and stimulate deadenylation in a partially redundant manner. Linear motifs from different RNA-binding factors that recruit CCR4-NOT to specific mRNAs via protein-protein interactions with CAF40 can inhibit bulk deadenylation. We reveal an additional layer of regulatory complexity to the human deadenylation machinery, which may prime it either for general or target-specific degradation.
CCR4-NOT 是一个保守的多蛋白复合物，主要通过信使 RNA 多聚 A 尾巴的缩短或去腺苷酸化来调节真核基因表达。在这里，我们重新构建了一个完整的、重组的人 CCR4-NOT 复合物。我们的重建策略允许严格的组成控制，以用纯化的组分变体测试机制假设。CCR4-NOT 比分离的外切核酸酶 CCR4a 和 CAF1 对 poly(A)更具活性和选择性，CAF1 在体外具有不同的去腺苷酸化谱。外切核酸酶至少需要三个保守的非酶模块（CAF40、NOT10：NOT11 或 NOT）中的两个，才能在 CCR4-NOT 中具有完全活性。CAF40 和 NOT10：NOT11 模块都能直接结合 RNA，并以部分冗余的方式刺激去腺苷酸化。通过与 CAF40 进行蛋白-蛋白相互作用将 CCR4-NOT 招募到特定 mRNA 的不同 RNA 结合因子的线性基序，可以抑制大量去腺苷酸化。我们揭示了人类去腺苷酸化机制的另一个调控复杂性，它可能使它要么对一般降解，要么对特定降解具有预备性。
