Ling Xiaobin, Golovenko Dmitrij, Gan Jianhua, Ma Jinbiao, Korostelev Andrei A, Fang Wenwen
RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA, USA.
State Key Laboratory of Genetics and Development of Complex Phenotypes, Department of Biochemistry and Biophysics, School of Life Sciences, Electron Microscopy Center, Fudan University, Shanghai, China.
Nature. 2025 Jun 16. doi: 10.1038/s41586-025-09262-x.
Long (>200 nucleotides) non-coding RNAs (lncRNAs) play important roles in diverse aspects of life. Over 20 classes of lncRNAs have been identified in bacteria and bacteriophages through comparative genomics analyses, but their biological functions remain largely unexplored. Due to the large sizes, the structural determinants of most lncRNAs also remain uncharacterized. Here we report the structures of two natural RNA nanocages formed by the lncRNA ROOL (rumen-originating, ornate, large) found in bacterial and phage genomes. ~2.9 Å cryo-electron microscopy (cryo-EM) structures reveal that ROOL RNAs form an octameric nanocage with a 28-nm diameter and 20-nm axial length, whose hollow inside features poorly ordered regions. The octamer is stabilized by numerous tertiary and quaternary interactions, including triple-strand A-minors that we propose to name "A-minor staples". The structure of an isolated ROOL monomer at ~3.2-Å resolution indicates that nanocage assembly involves a strand-swapping mechanism resulting in quaternary kissing loops. Finally, we show that ROOL RNA fused to an RNA aptamer, tRNA, or microRNA retains its structure forming a nanocage with radially displayed cargos. Our findings therefore may enable the engineering of novel RNA nanocages as delivery vehicles for research and therapeutic applications.
长链（>200个核苷酸）非编码RNA（lncRNAs）在生命的各个方面发挥着重要作用。通过比较基因组学分析，在细菌和噬菌体中已鉴定出20多种lncRNAs，但它们的生物学功能在很大程度上仍未被探索。由于lncRNAs尺寸较大，大多数lncRNAs的结构决定因素也尚未明确。在这里，我们报道了在细菌和噬菌体基因组中发现的由lncRNA ROOL（源于瘤胃、华丽、大型）形成的两种天然RNA纳米笼的结构。约2.9 Å的冷冻电子显微镜（cryo-EM）结构表明，ROOL RNA形成了一个直径为28 nm、轴向长度为20 nm的八聚体纳米笼，其内部中空区域特征为无序区域。八聚体通过众多三级和四级相互作用得以稳定，包括我们提议命名为“A-小钉”的三链A-小沟相互作用。约3.2 Å分辨率下分离的ROOL单体结构表明，纳米笼组装涉及一种链交换机制，导致四级亲吻环。最后，我们表明，与RNA适体、tRNA或微小RNA融合的ROOL RNA保留其结构，形成带有径向展示货物的纳米笼。因此，我们的发现可能使新型RNA纳米笼的工程设计成为用于研究和治疗应用的递送载体。
