Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland.
Department of Biochemistry, University of Zürich, Zürich, Switzerland.
EMBO J. 2018 Mar 15;37(6). doi: 10.15252/embj.201797089. Epub 2018 Feb 15.
The accurate cleavage of pre-micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA-induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N-terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single-molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence-specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo-symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer-TRBP-siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer-mediated cleavage accuracy by binding the dsRNA region of the pre-miRNA during Dicer cleavage.
Dicer 准确切割前 microRNA(miRNA) 和 miRNA/siRNA 引导链选择是形成 RNA 诱导沉默复合物 (RISC) 的重要步骤。Dicer 结合伴侣 TRBP 在这些过程中的作用仍知之甚少。在这里，我们使用 NMR、EPR 和单分子光谱法解决了与功能不对称 siRNA 结合的 TRBP 的两个 N 端双链 RNA 结合结构域 (dsRBD) 的溶液结构。我们发现 dsRBD 对 siRNA 的识别不是序列特异性的，而是依赖于 RNA 形状。两个 dsRBD 可以交换它们的结合位点，产生两个同样多的、拟对称的复合物，表明 TRBP 不是 siRNA 不对称性的主要传感器。使用我们的结构来模拟 Dicer-TRBP-siRNA 三元复合物，我们表明 TRBP 的 dsRBD 和 Dicer 的 RNase III 结构域在相反的两侧结合一个典型的 19 碱基对 siRNA，支持一种机制，即 TRBP 通过在 Dicer 切割期间结合 pre-miRNA 的 dsRNA 区域来影响 Dicer 介导的切割准确性。
