MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which are subsequently cleaved by Dicer to generate mature miRNAs. Instead of being cleaved by two different enzymes, both cleavages in plants are performed by Dicer-like 1 (DCL1). With a similar domain architecture as human Dicer, it is mysterious how DCL1 recognizes pri-miRNAs and performs two cleavages sequentially. Here, we report the single-particle cryo-electron microscopy structures of Arabidopsis DCL1 complexed with a pri-miRNA and a pre-miRNA, respectively, in cleavage-competent states. These structures uncover the plasticity of the PAZ domain, which is critical for the recognition of both pri-miRNA and pre-miRNA. These structures suggest that the helicase module serves as an engine that transfers the substrate between two sequential cleavage events. This study lays a foundation for dissecting the regulation mechanism of miRNA biogenesis in plants and provides insights into the dicing state of human Dicer.

微小 RNA (miRNA) 是短的非编码 RNA，通过直接结合其 mRNA 来抑制靶基因的表达。在动物中，pri-miRNA 被 Drosha 切割以产生 pre-miRNA，随后被 Dicer 切割以产生成熟的 miRNA。植物中的两种切割均由 Dicer-like 1 (DCL1) 执行，而不是被两种不同的酶切割。由于具有与人类 Dicer 相似的结构域结构，DCL1 如何识别 pri-miRNA 并依次执行两次切割仍然是个谜。在这里，我们报告了拟南芥的单粒子冷冻电子显微镜结构DCL1 在可切割状态下分别与 pri-miRNA 和 pre-miRNA 复合。这些结构揭示了 PAZ 结构域的可塑性，这对于识别 pri-miRNA 和 pre-miRNA 都至关重要。这些结构表明解旋酶模块充当在两个连续的切割事件之间转移底物的引擎。本研究为剖析植物中 miRNA 生物发生的调控机制奠定了基础，并提供了对人类 Dicer 切割状态的见解。