Kristoffersen Emil L, McRae Ewan K S, Sørensen Niels R, Holliger Philipp, Andersen Ebbe S
Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus, Denmark.
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf057.
The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have not been explored. Here, we have used cryogenic electron microscopy (cryo-EM) to characterize products and intermediates relevant for RCS replication. We find that these form an unexpectedly diverse group of RNA nanostructures. The main structural motif observed is a fully hybridized dimeric complex composed of two scRNAs and their complement strands resolved to 5.3 Å. Cryo-EM also reveals higher-order dimer filaments and dimer assembly intermediates, suggesting an assembly mechanism for the observed complexes. We show that the dimer complexes are stable and inhibit RNA-catalyzed RCS but can be reactivated by addition of more scRNA templates. We propose dimer formation as a general property of RCS replication and speculate that dimers might have benefited a primordial RNA genetic system by providing a stable ''storage'' form for RNA replication products and by coordinated RNA replication on both scRNA template strands.
RNA世界假说得到了RNA聚合酶核酶的发现的支持，这种酶可以在不同的RNA模板上进行RNA催化的RNA复制过程。最近，已证明在小环形RNA（scRNA）模板上可进行RNA催化的滚环合成（RCS）。然而，scRNA复制及其产物和中间体的结构和动态特性尚未得到探索。在这里，我们使用低温电子显微镜（cryo-EM）来表征与RCS复制相关的产物和中间体。我们发现这些形成了一组出乎意料的多样化的RNA纳米结构。观察到的主要结构基序是由两条scRNA及其互补链组成的完全杂交的二聚体复合物，分辨率达到5.3 Å。低温电子显微镜还揭示了高阶二聚体细丝和二聚体组装中间体，这表明了所观察到的复合物的组装机制。我们表明二聚体复合物是稳定的，并抑制RNA催化的RCS，但可以通过添加更多的scRNA模板重新激活。我们提出二聚体形成是RCS复制的一个普遍特性，并推测二聚体可能通过为RNA复制产物提供一种稳定的“储存”形式以及通过在两条scRNA模板链上协调RNA复制，使原始RNA遗传系统受益。
