Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [nanoPARE]

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localizes with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting its activity in reprograming gene expression during stress. Collectively, our work shed new light on the impact of high temperatures on the main effector of RNA silencing in plants. RNAseq from Arabidopsis seedlings under control, heat stress, and recovery conditions. Three biological replicates each.

在拟南芥（Arabidopsis thaliana）中，ARGONAUTE1（AGO1）在微小RNA（microRNA，miRNA）与小干扰RNA（small interfering RNA，siRNA）介导的基因沉默过程中发挥核心作用。细胞核内的AGO1可结合miRNA并被转运至细胞质，在粗面内质网（rough ER）处介导miRNA依赖的翻译抑制、mRNA剪切以及相位依赖性siRNA的生物发生。上述相位依赖性siRNA及其他细胞质siRNA均可结合细胞质中的AGO1，但该结合过程发生于何种亚细胞区域目前仍不明确。此外，胁迫对AGO1亚细胞定位的影响仍有待阐明。本研究发现，热胁迫（heat stress，HS）可促进AGO1蛋白积累，且积累的AGO1与siRNA小体（siRNA bodies）及应激颗粒（stress granules，SGs）的组分共定位。AGO1在热胁迫过程中高效形成凝聚体并不依赖于siRNA小体的关键组分SGS3。相反，本研究发现，AGO1中尚未得到充分研究的、包含朊蛋白样结构域（prion-like domain）的N端聚谷氨酰胺结构域（Poly-Q domain）足以介导相分离（phase separation）。此外，仅1小时的热胁迫处理仅会适度影响AGO1结合miRNA及其靶标剪切的能力，这表明AGO1在凝聚体中的定位对其起到保护作用，而非促进其在胁迫过程中重编程基因表达的活性。综上，本研究为高温对植物RNA沉默核心效应因子的影响提供了新的研究视角。本研究的RNA测序（RNA-seq）数据来自于对照、热胁迫及恢复培养条件下的拟南芥幼苗，每组均设置3次生物学重复。