In plants, HEN1-facilitated methylation at 3′ end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1’s methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

中文翻译：

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通过被子植物和苔藓植物中 HEN1 的功能比较研究病毒抑制剂 HC-Pro 抑制小 RNA 甲基化

在植物中，HEN1 促进的 3' 末端核糖甲基化是小 RNA (sRNA) 生物发生的关键步骤。经过充分研究的拟南芥 HEN1 (AtHEN1) 突变体hen1-1显示出有缺陷的发育表型，表明 sRNA 甲基化的重要性。此外，Marchantia polymorpha已被鉴定为具有HEN1直系同源基因（Mp HEN1); 然而，它的功能仍然深不可测。我们的体内和体外数据显示 MpHEN1 活性与 AtHEN1 相当，并且它们对双链体 microRNA (miRNA) 的底物特异性保持一致。此外，系统发育树和多重比对突出了植物中 HEN1 家族的保守分子进化。萝卜花叶病毒 (TuMV) 的 P1/HC-Pro 是一种已知的 RNA 沉默抑制因子，可抑制 sRNA 的 HEN1 甲基化。在这里，我们报告 HC-Pro 通过 FRNK 基序与 AtHEN1 物理结合，抑制 HEN1 的甲基化活性。此外，体外 EMSA 数据表明 TuMV 的 GST-HC-Pro 缺乏 sRNA 双链体结合能力。令人惊讶的是，HC-Pro 还以剂量依赖性方式抑制 MpHEN1 活性，这表明 HC-Pro 和 MpHEN1 之间也可能存在相互作用。