RNA interference (RNAi) plays important roles in organism development through post-transcriptional regulation of specific target mRNAs. Target specificity is largely controlled by base-pair complementarity between micro-RNA (miRNA) regulatory elements and short regions of the target mRNA. The pattern of miRNA production in a cell interacts with the cell's mRNA transcriptome to generate a specific network of post-transcriptional regulation that can play critical roles in cellular metabolism, differentiation, tissue/organ development and developmental timing. In plants, miRNA production is orchestrated in the nucleus by a suite of proteins that control transcription of the pri-miRNA gene, post-transcriptional processing and nuclear export of the mature miRNA. In the model plant, Arabidopsis thaliana, post-transcriptional processing of miRNAs is controlled by a pair of physically-interacting proteins, HYL1 and DCL1. However, the evolutionary history of the HYL1-DCL1 interaction is unknown, as is its structural basis. Here we use ancestral sequence reconstruction and functional characterization of ancestral HYL1 in vitro and in vivo 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 very early in plant evolutionary history, 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 A. thaliana 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'; its role in generating developmental novelty is probably related to the relatively rapid evolution of miRNA genes.

中文翻译：

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古老，保守的开发工具箱，用于控制陆地植物转录后基因调控的直接分子证据

RNA干扰（RNAi）通过转录后特定靶标mRNA的调控在生物体发育中发挥重要作用。靶标特异性在很大程度上由microRNA（miRNA）调节元件与靶标mRNA短区域之间的碱基对互补性控制。细胞中miRNA产生的模式与细胞的mRNA转录组相互作用，生成转录后调控的特定网络，该网络可在细胞代谢，分化，组织/器官发育和发育时机中发挥关键作用。在植物中，miRNA的产生是通过控制pri-miRNA基因转录，转录后加工和成熟miRNA核输出的一组蛋白质在细胞核中进行协调的。在模型植物拟南芥中，miRNA的转录后加工受一对物理相互作用蛋白HYL1和DCL1的控制。但是，HYL1-DCL1相互作用的进化历史及其结构基础尚不清楚。在这里，我们在体外和体内使用祖先序列的重建和祖先HYL1的功能表征，以更好地了解HYL1-DCL1相互作用的起源和进化及其对miRNA生产和植物发育的影响。我们发现祖先植物HYL1在植物进化史的早期就对双链RNA（dsRNA）及其DCL1伴侣都形成了高亲和力，在种子植物中的苔藓发散之前（〜500 Ma），这些高亲和力相互作用仍然存在在整个植物进化史上基本上都是保守的。结构建模和分子结合实验表明，HYL1中的两个双链RNA结合基序（DSRM）中的第二个可能与两个C端DCL1 DSRM的第一个紧密相互作用，以介导有效产生miRNA所必需的HYL1-DCL1物理相互作用。拟南芥中近200株Ma-old祖先开花植物HYL1的转基因表达足以挽救HYL1-敲除破坏的植物发育的许多关键方面，并恢复了近乎原生的miRNA产生，这表明HYL1-DCL1的功能伙伴关系起源于很早，在整个陆地植物的进化史中都得到了高度的保护。总体而言，我们的结果与一个模型相吻合，在该模型中，基于miRNA的基因调控作为保守植物“发育工具包”的一部分而发展。