RNA-directed DNA methylation in plants is guided by 24-nt siRNAs generated in parallel with 23-nt RNAs of unknown function. We show that 23-nt RNAs function as passenger strands during 24-nt siRNA incorporation into AGO4. The 23-nt RNAs are then sliced into 11- and 12-nt fragments, with 12-nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21–24 nt, with any 5′-terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. In vivo, RdDM target locus RNAs that copurify with AGO4 also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RNA-directed DNA methylation.

中文翻译：

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AGO4 在 RNA 指导的 DNA 甲基化中的酶促反应：siRNA 双链加载、过客链消除、目标 RNA 切片和切片目标保留

植物中 RNA 指导的 DNA 甲基化由与未知功能的 23-nt RNA 平行生成的 24-nt siRNA 指导。我们显示 23-nt RNA 在 24-nt siRNA 掺入 AGO4 期间充当过客链。然后将 23-nt RNA 切成 11-nt 和 12-nt 片段，其中 12-nt 片段仍与 AGO4 相关。用重组 AGO4 和合成 RNA 概括的切片显示，21-24 nt 的 siRNA，具有任何 5'-末端核苷酸，可以指导切片，然后切片的 RNA 被 AGO4 保留。在体内，与 AGO4 共纯化的 RdDM 靶基因座 RNA 也显示出切片序列特征。比较表达切片能力和切片缺陷 AGO4 的植物表明，切片提高了几乎所有 RdDM 位点的胞嘧啶甲基化水平。