Direct molecular evidence for an ancient, conserved developmental toolkit controlling post-transcriptional gene regulation in land plants

In plants, miRNA production is orchestrated by a suite of proteins that control transcription of the pri-miRNA gene, post-transcriptional processing and nuclear export of the mature miRNA. Post-transcriptional processing of miRNAs is controlled by a pair of physically-interacting proteins, HYL1 and DCL1. However, the evolutionary history and structural basis of the HYL1-DCL1 interaction is unknown. Here we use ancestral sequence reconstruction and functional characterization of ancestral HYL1 <em>in vitro</em> and in <em>Arabidopsis thaliana </em>to better understand the origin and evolution of the HYL1-DCL1 interaction and its impact on miRNA production and plant development. We found the ancestral plant HYL1 evolved high affinity for both double-stranded RNA (dsRNA) and its DCL1 partner before the divergence of mosses from seed plants (~500 Ma), and these high-affinity interactions remained largely conserved throughout plant evolutionary history. Structural modeling and molecular binding experiments suggest that the second of two double-stranded RNA-binding motifs (DSRMs) in HYL1 may interact tightly with the first of two C-terminal DCL1 DSRMs to mediate the HYL1-DCL1 physical interaction necessary for efficient miRNA production. Transgenic expression of the nearly 200 Ma-old ancestral flowering-plant HYL1 in <em>A. thaliana</em> was sufficient to rescue many key aspects of plant development disrupted by HYL1^- knockout and restored near-native miRNA production, suggesting that the functional partnership of HYL1-DCL1 originated very early in and was strongly conserved throughout the evolutionary history of terrestrial plants. Overall, our results are consistent with a model in which miRNA-based gene regulation evolved as part of a conserved plant ‘developmental toolkit’.