Structural Cell Biology Department, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried/Munich, D-82152 Germany and Cellular Biochemistry Department, Max Planck Institute of Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany.
Nucleic Acids Res. 2013 Sep;41(17):8377-90. doi: 10.1093/nar/gkt600. Epub 2013 Jul 12.
Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5' cap structure is a crucial step that commits deadenylated mRNAs to 5'-to-3' degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking-mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.
真核生物 mRNA 的翻译抑制和加尾作用会导致转录本被隔离在非翻译池或降解。去除 5'帽结构是一个关键步骤，它使加尾的 mRNA 能够进行 5'到 3'降解。Pat1、Edc3 和 DEAD-box 蛋白 Dhh1 是进化上保守的因子，已知它们参与翻译抑制和脱帽作用，但它们之间的相互作用目前尚不清楚。我们报告了酵母 Dhh1 与 Pat1 的 N 端结构域结合的 2.8 Å 分辨率结构。该结构显示了 Pat1 如何包裹 Dhh1 的 C 端 RecA 结构域，对接在苯丙氨酸-天冬氨酸-苯丙氨酸（FDF）结合位点上。Dhh1 的 FDF 结合位点也识别 Edc3，这解释了为什么 Pat1 和 Edc3 在 Dhh1 上的结合是相互排斥的事件。通过共免疫沉淀测定和基于结构的突变体，我们证明了 Dhh1-Pat1 识别模式在人类中是保守的。Pat1 和 Edc3 也干扰和竞争 Dhh1 的 RNA 结合特性。通过交联质谱法在 Dhh1 上绘制 RNA 结合位点，显示在 C 端 RecA 结构域周围有一个大的 RNA 结合表面，包括 FDF 结合口袋。结果表明，在 Pat1 和 Edc3 结合后，Dhh1 包含的信使核糖核蛋白颗粒可能会发生重塑的模型。
