Long noncoding RNAs (lncRNAs) are increasingly appreciated for their important functions in mammalian cells. However, how their functional capacities are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA-binding proteins, but the structural principles that underlie this mode of regulation are unknown. The NORAD lncRNA is a known decoy for Pumilio proteins, which modulate the translation and stability of hundreds of messenger RNAs and, consequently, a regulator of genomic stability and aging. Here we probed the RNA structure and long-range RNA–RNA interactions formed by human NORAD inside cells under different stressful conditions. We discovered a highly modular structure consisting of well-defined domains that contribute independently to NORAD function. Following arsenite stress, most structural domains undergo relaxation and form interactions with other RNAs that are targeted to stress granules. We further revealed a unique structural organization that spatially clusters the multiple Pumilio binding sites along NORAD and consequently contributes to the derepression of Pumilio targets. We then applied these structural principles to design an effective artificial decoy for the let-7 microRNA. Our work demonstrates how the sequence of a lncRNA spatially clusters its function into separated domains and how structural principles can be employed for the rational design of lncRNAs with desired activities.

长非编码 RNA (lncRNA) 因其在哺乳动物细胞中的重要功能而日益受到重视。然而，它们的功能能力如何在其序列中编码并在其结构中体现仍然很大程度上未知。一些 lncRNA 结合并调节 RNA 结合蛋白的可用性，但这种调节模式的结构原理尚不清楚。 NORAD lncRNA 是 Pumilio 蛋白的已知诱饵，它调节数百种信使 RNA 的翻译和稳定性，因此是基因组稳定性和衰老的调节剂。在这里，我们探讨了不同应激条件下细胞内人类 NORAD 形成的 RNA 结构和长程 RNA-RNA 相互作用。我们发现了一种高度模块化的结构，由明确定义的域组成，这些域独立地为 NORAD 功能做出贡献。亚砷酸盐应激后，大多数结构域都会松弛并与其他针对应激颗粒的 RNA 形成相互作用。我们进一步揭示了一种独特的结构组织，该结构组织沿着 NORAD 在空间上聚集多个 Pumilio 结合位点，从而有助于 Pumilio 靶点的去抑制。然后，我们应用这些结构原理为 let-7 microRNA 设计有效的人工诱饵。我们的工作展示了 lncRNA 的序列如何在空间上将其功能聚集成独立的域，以及如何利用结构原理来合理设计具有所需活性的 lncRNA。