Department of Biology, Indiana University, Bloomington 47405, Indiana, USA.
Donald Danforth Plant Science Center, St Louis 63132, Missouri, USA.
Plant Cell. 2022 Apr 26;34(5):1863-1881. doi: 10.1093/plcell/koac043.
Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.
先前，我们已经表明，从拟南芥叶片中纯化的质外体洗脱液 (AWF) 含有小 RNA (sRNA)。为了研究这些 sRNA 是否被包裹在细胞外囊泡 (EV) 内，我们用蛋白酶胰蛋白酶和 RNase A 处理从拟南芥叶片中分离的 EV，这两种酶应该降解位于 EV 外但不位于 EV 内的 RNA。这些分析表明，质外体 RNA 主要位于细胞外，与蛋白质相关。对这些细胞外 RNA (exRNA) 的进一步分析表明，它们包括 sRNA 和长非编码 RNA (lncRNA)，包括环状 RNA (circRNA)。我们还发现 exRNA 高度富集在后转录修饰 N6-甲基腺嘌呤 (m6A) 中。与此一致，我们在 AWF 中鉴定了一种假定的 m6A 结合蛋白，甘氨酸丰富 RNA 结合蛋白 7 (GRP7)，以及 sRNA 结合蛋白 ARGONAUTE2 (AGO2)。这两种蛋白质与包括 circRNA 在内的 lncRNA 共免疫沉淀。GRP7 或 AGO2 的突变导致 AWF 中 sRNA 和 lncRNA 含量的变化，表明这些蛋白质有助于 exRNA 的分泌和/或稳定。我们提出，位于 EV 外的 exRNA 介导宿主诱导的基因沉默，而不是位于 EV 内的 RNA。
