HEN1 甲基化能力對於植物 RNA 靜默影響層次研究

Abstract

RNA靜默 (RNA silencing) 是調控基因表達的重要機制，主要透過小RNA (如siRNA和miRNA) 來調節目標mRNA的切割或翻譯抑制。HUA ENHANCER 1 (HEN1) 作為2'-O-甲基轉移酶，負責對小RNA的3'端進行甲基化修飾，以保護其免受核酸酶降解並維持穩定性。當HEN1失去活性時會產生未甲基化的miRNA，隨後HESO1會對其進行尿苷化修飾，使其降解。P1/HC-ProTu作為RNA靜默的病毒抑制子 (VSRs)，其功能會抑制HEN1結合miRNA的活性，進而影響RNA靜默機制。本研究探討了HEN1體外與體內甲基化活性對植物基因調控及RNA靜默能力的影響。我們成功構建了可誘導表達的HESO1轉基因植物 (HA-HESO1/P1/HC-ProTu/heso1-1和HESO1-HA/P1/HC-ProTu/heso1-1)，為進一步研究HESO1與AGO1相互作用奠定基礎。透過體外生化實驗證實，his-AtHEN1D719N完全失去甲基化miRNA的能力，直接證明了D719N突變對RNA靜默功能的關鍵影響。值得注意的是，地錢 (Marchantia polymorpha) MpHEN1中相同保守位點的突變 (D760N) 仍保留部分甲基化活性，顯示不同物種間蛋白質功能演化的差異。轉錄組分析顯示，hen1-8突變體和P1/HC-ProTu在基因表達模式上表現出相似性。基因-基因網絡分析發現76個共同基因，其中11個基因與光信號傳導相關，包括生物鐘核心組件CCA1、LHY和PRR5等，進一步揭示RNA silencing可能與其他機制的相關性。降解組分析進一步揭示了HEN1、HESO1和P1/HC-ProTu在調節RNA靜默中的複雜相互作用。本研究為理解HEN1甲基化活性在植物發育和環境適應中的分子機制提供了重要見解，且首次在體外實驗證實了AtHEN1D719N的甲基化功能喪失，並藉由基因-基因網絡分析為RNA靜默機制的研究開闢了新方向。

RNA silencing is a crucial regulatory mechanism for controlling gene expression, primarily mediated by small RNAs such as short interfering RNAs (siRNAs) and microRNAs (miRNAs), which regulate target mRNA cleavage or translational repression. HUA ENHANCER 1 (HEN1), functioning as a 2'-O-methyltransferase, is responsible for methylating the 3' termini of small RNAs to protect them from nuclease degradation and maintain their stability. Loss of HEN1 activity results in the production of unmethylated miRNAs, which are subsequently uridylated by HESO1 and targeted for degradation. P1/HC-ProTu, serving as a viral suppressor of RNA silencing (VSR), functions by inhibiting the methyltransferase (MTase) activity of HEN1 to bind miRNAs, thereby disrupting RNA silencing mechanisms. This study examines the impact of HEN1 MTase activity, both in vitro and in vivo, on plant gene regulation and RNA silencing capacity. We successfully constructed inducible HESO1 transgenic plants (HA-HESO1/P1/HC-ProTu/heso1-1 and HESO1-HA/P1/HC-ProTu/heso1-1 plants) and established a foundation for further investigation of HESO1-mediated autophagic AGO1 degradation. Moreover, through in vitro methylation activity assay, we confirmed that his-AtHEN1D719N (the mutant form of AtHEN1) completely loses its ability to methylate miRNAs, directly demonstrating the critical impact of the D719N mutation on RNA silencing function. Notably, the corresponding conserved site mutation (D760N) in Marchantia polymorpha HEN1 (MpHEN1) retains partial methylation activity, revealing evolutionary differences in protein function between species. Transcriptomic analysis revealed similarities in gene expression patterns between hen1-8 mutant and P1/HC-ProTu plants. Gene-to-gene network analysis identified 76 common genes, of which 11 are associated with light signaling pathways, including core circadian clock components CCA1, LHY, and PRR5. This further reveals potential correlations between RNA silencing and other regulatory mechanisms. Degradome analysis further elucidated the complex interactions among HEN1, HESO1, and P1/HC-ProTu in the regulation of RNA silencing. This study offers valuable insights into the molecular mechanisms underlying HEN1 methylation activity in plant development and environmental adaptation. We present the first in vitro experimental evidence confirming the loss of methylation function in AtHEN1D719N and establish new research directions for RNA silencing mechanisms through gene-to-gene network analysis.