MicroRNAs (miRNAs) are produced from highly structured primary transcripts (pri-miRNAs) and regulate numerous biological processes in eukaryotes. Due to the extreme heterogeneity of these structures, the initial processing sites of plant pri-miRNAs and the structural rules that determine their processing have been predicted for many miRNAs but remain elusive for others. Here we used semi-active DCL1 mutants and advanced degradome-sequencing strategies to accurately identify the initial processing sites for 147 of 326 previously annotated Arabidopsis miRNAs and to illustrate their associated pri-miRNA cleavage patterns. Elucidating the in vivo RNA secondary structures of 73 pri-miRNAs revealed that about 95% of them differ from in silico predictions, and that the revised structures offer clearer interpretation of the processing sites and patterns. Finally, DCL1 partners Serrate and HYL1 could synergistically and independently impact processing patterns and in vivo RNA secondary structures of pri-miRNAs. Together, our work sheds light on the precise processing mechanisms of plant pri-miRNAs.

MicroRNA (miRNA) 由高度结构化的初级转录物 (pri-miRNA) 产生，调节真核生物中的许多生物过程。由于这些结构的极端异质性，植物 pri-miRNA 的初始加工位点以及决定其加工的结构规则对于许多 miRNA 来说已经被预测，但对于其他 miRNA 来说仍然难以捉摸。在这里，我们使用半活性 DCL1 突变体和先进的降解组测序策略来准确识别 326 个先前注释的拟南芥miRNA 中的 147 个的初始加工位点，并说明它们相关的 pri-miRNA 切割模式。阐明 73 个 pri-miRNA 的体内 RNA 二级结构表明，其中约 95% 与计算机预测不同，并且修改后的结构为加工位点和模式提供了更清晰的解释。最后，DCL1 伙伴 Serrate 和 HYL1 可以协同且独立地影响 pri-miRNA 的加工模式和体内 RNA 二级结构。我们的工作共同揭示了植物 pri-miRNA 的精确加工机制。