RNA G-quadruplexes (rG4s) are noncanonical RNA secondary structures formed by guanine (G)-rich sequences. These complexes play important regulatory roles in both animals and plants through their structural dynamics and are closely related to human diseases and plant growth, development, and adaption. Thus, studying the structural dynamics of rG4s is fundamentally important; however, their folding pathways and their unfolding by specialized helicases are not well understood. In addition, no plant rG4-specialized helicases have been identified. Here, using single-molecule FRET, we experimentally elucidated for the first time the folding pathway and intermediates, including a G-hairpin and G-triplex. In addition, using proteomics screening and microscale thermophoresis, we identified and validated five rG4-specialized helicases in Arabidopsis thaliana. Furthermore, DExH1, the ortholog of the famous human rG4 helicase RHAU/DHX36, stood out for its robust rG4 unwinding ability. Taken together, these results shed light on the structural dynamics of plant rG4s.

RNA G-四链体 (rG4s) 是由富含鸟嘌呤 (G) 的序列形成的非经典 RNA 二级结构。这些复合物通过其结构动力学在动植物中发挥重要的调节作用，与人类疾病和植物的生长、发育和适应密切相关。因此，研究 rG4s 的结构动力学至关重要。然而，它们的折叠路径和它们通过专门的解旋酶展开的过程还不是很清楚。此外，尚未鉴定出植物 rG4 特化的解旋酶。在这里，我们使用单分子 FRET，首次通过实验阐明了折叠途径和中间体，包括 G-发夹和 G-三链体。此外，使用蛋白质组学筛选和微量热泳，我们在拟南芥。此外，著名的人类 rG4 解旋酶 RHAU/DHX36 的直系同源物 DExH1 因其强大的 rG4 解旋能力而脱颖而出。总之，这些结果揭示了植物 rG4s 的结构动力学。