Max F. Perutz Laboratories, University of Vienna, Department of Microbiology, Immunbiology and Genetics, Vienna, Austria.
PLoS One. 2013 Aug 19;8(8):e71860. doi: 10.1371/journal.pone.0071860. eCollection 2013.
Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.
动物 miRNAs 通常通过与靶 mRNAs 结合来介导 mRNA 的降解和/或翻译抑制。miRNA 介导的 mRNA 降解的关键因素是 miRNA 效应复合物（AGO1 和 GW182）和一般 mRNA 降解机制（腺苷酸化和去帽酶）的组成部分。靶 mRNAs 去腺苷酸化所需的 CCR4-NOT1 复合物被直接招募到 miRNA 效应复合物。然而，尚不清楚以下去帽步骤是否仅仅是 miRNA 效应复合物独立发生的腺苷酸化的结果，或者例如去帽激活剂是否可以被招募到 miRNA 效应复合物。在本研究中，我们在果蝇细胞中进行了分裂亲和力纯化，并提供了去帽激活剂 HPat 与 miRNA 效应复合物相互作用的证据。此外，在对各种 mRNA 降解因子的敲低分析中，我们证明了 NOT1 对这种相互作用的重要性。这表明去腺苷酸化和/或 NOT1 蛋白的募集先于 HPat 与 miRNA 效应复合物的结合。由于 HPat 偶联了去腺苷酸化和去帽，因此 HPat 被招募到 miRNA 效应复合物提供了一种将 mRNA 靶标用于降解的机制。
