The interaction of mRNA and regulatory proteins is critical for posttranscriptional control. For proper function, these interactions, as well as the involved protein and RNA structures, are highly dynamic, and thus, mechanistic insights from structural biology are challenging to obtain. In this study, we employ a multifaceted approach combining single-molecule force spectroscopy (SMFS) with NMR spectroscopy to analyze the concerted interaction of the two RNA-binding interfaces (A-site and B-site) of the immunoregulatory protein Roquin’s ROQ domain with the 3’ untranslated region (UTR) of the Ox40 mRNA. This 3’UTR contains two specific hairpin structures termed constitutive and alternative decay elements (CDE, ADE), which mediate mRNA degradation through Roquin binding. Our single-molecule experiments reveal that the CDE folds cooperatively, while ADE folding involves at least three on-pathway and three off-pathway intermediates. Using an integrated microfluidics setup, we extract binding kinetics to Roquin in real time. Supported by NMR data, we find opposing effects of the two Roquin subdomains on distinct regions of the ADE: While the A-site interacts strongly with the folded apical stem-loop, we find that the B-site has a distinct destabilizing effect on the central stem of the ADE owed to single-strand RNA binding. We propose that RNA-motif nature and Roquin A- and B-sites jointly steer mRNA decay with context-encoded specificity, and we suggest plasticity of stem structures as key determinant for Roquin–RNA complex formation. The unique combination of NMR and SMFS uncovers a mechanism of a dual-function RNA-binding domain, offering a model for target RNA recognition by Roquin.

中文翻译：

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Roquin 通过其两个 ROQ 结构域结合位点对 RNA 茎环稳定性表现出相反的影响


mRNA 和调节蛋白的相互作用对于转录后控制至关重要。为了实现正常功能，这些相互作用以及所涉及的蛋白质和 RNA 结构是高度动态的，因此，从结构生物学中获得机制见解是具有挑战性的。在这项研究中，我们采用一种将单分子力谱 （SMFS） 与 NMR 波谱相结合的多方面方法来分析免疫调节蛋白 Roquin 的 ROQ 结构域的两个 RNA 结合界面（A 位点和 B 位点）与 Ox40 mRNA 的 3' 非翻译区 （UTR） 的协同相互作用。该 3'UTR 包含两个特定的发夹结构，称为组成型和选择性衰变元件 （CDE、ADE），它们通过 Roquin 结合介导 mRNA 降解。我们的单分子实验表明，CDE 协同折叠，而 ADE 折叠涉及至少 3 个通路上和 3 个非通路中间体。使用集成的微流体设置，我们实时提取与 Roquin 的结合动力学。在 NMR 数据的支持下，我们发现两个 Roquin 亚结构域对 ADE 的不同区域有相反的影响：虽然 A 位点与折叠的顶端茎环强烈相互作用，但我们发现 B 位点对 ADE 的中央茎具有明显的不稳定作用，这归因于单链 RNA 结合。我们提出 RNA 基序性质和 Roquin A 和 B 位点以上下文编码的特异性共同引导 mRNA 衰变，并且我们建议茎结构的可塑性是 Roquin-RNA 复合物形成的关键决定因素。NMR 和 SMFS 的独特组合揭示了双功能 RNA 结合结构域的机制，为 Roquin 识别靶 RNA 提供了模型。