Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA.
Genes Dev. 2023 Feb 1;37(3-4):103-118. doi: 10.1101/gad.350240.122. Epub 2023 Feb 6.
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.
在植物中，RNA 指导的 DNA 甲基化是由与功能未知的 23nt RNA 平行产生的 24nt siRNA 指导的。我们表明，23nt RNA 在 24nt siRNA 整合到 AGO4 中时作为过客链发挥作用。然后，23nt RNA 被切成 11nt 和 12nt 片段，12nt 片段仍然与 AGO4 相关联。用重组 AGO4 和合成 RNA 进行的切割再现表明，具有任何 5'-末端核苷酸的 21-24nt 的 siRNA 可以指导切割，然后切割的 RNA 被 AGO4 保留。在体内，与 AGO4 共纯化的 RdDM 靶标 RNA 也显示出切割的序列特征。比较表达有切割能力和无切割能力的 AGO4 的植物表明，切割几乎可以提高所有 RdDM 基因座的胞嘧啶甲基化水平。我们提出，siRNA 过客链的消除和 AGO4 与切割的靶 RNA 的连接是 AGO4 切割增强 RNA 指导的 DNA 甲基化的两种不同方式。
