Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, which is de-regulated in various cancers, in human Argonaute-2 (hAgo2), the effector protein in RISC. This analysis employs guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced Magic Angle Spinning (MAS) NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and subsequently bound to SIRT1 mRNA compared to the free miR-34a or the free mRNA:miR duplex. The C8–C1′ correlation provided a nucleotide-specific distribution of C2′- and C3′-endo sugar puckering, revealing the capture of diverse dynamic conformations upon freezing. Predominantly C3′-endo puckering was observed for the seed region, while C2′-endo conformation was found in the central region, with a mixture of both conformations elsewhere. These observations provide insights into the molecular dynamics underlying miR-mediated mRNA regulation and demonstrate that experiments conducted under cryogenic conditions, such as at 90 K, can capture and reveal frozen dynamic states, using methods like DNP-enhanced MAS NMR or Cryo-Electron Microscopy.

中文翻译：

---

通过动态核极化增强魔角旋转 NMR 阐明人 Argonaute-2 中的 microRNA-34a 组织


了解 microRNA （miR） 对 mRNA 的调控依赖于对 RNA 诱导沉默复合物 （RISC） 的结构理解。在这里，我们阐明了 miR-34a 的结构组织，该组织在各种癌症中失调，在人 Argonaute-2 （hAgo2） 中，RISC 中的效应蛋白。该分析采用鸟苷特异性同位素标记和动态核极化 （DNP） 增强的魔角旋转 （MAS） NMR。同核相关实验显示，与游离 miR-34a 或游离 mRNA：miR 双链体相比，miR-34a 的非 A 型螺旋构象在掺入 hAgo2 时增加，随后与 SIRT1 mRNA 结合。C8-C1′ 相关性提供了 C2′-和 C3′-内切糖缩皱的核苷酸特异性分布，揭示了冷冻时不同动态构象的捕获。主要在种子区域观察到 C3′-endo 缩皱，而在中央区域发现 C2′-endo 构象，在其他地方混合了两种构象。这些观察结果提供了对 miR 介导的 mRNA 调控的分子动力学的见解，并表明在低温条件下（例如 90 K）进行的实验可以使用 DNP 增强型 MAS NMR 或冷冻电子显微镜等方法捕获和揭示冻结的动态状态。