Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [nanoPARE]. 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. Overall design: 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microRNA（miRNA）与small interfering RNA（siRNA）介导的RNA沉默过程中发挥核心作用。细胞核内的AGO1会结合miRNA并被转运至细胞溶质，随后与粗面内质网（rough ER）结合，执行miRNA介导的翻译抑制、mRNA剪切以及阶段性siRNA的生物发生。上述阶段性siRNA与其他胞质siRNA均可结合至胞质AGO1，但该结合过程发生于何种亚细胞区室目前尚不明确。此外，应激对AGO1亚细胞定位的影响仍有待阐明。

本研究显示，热应激（heat stress, HS）可促进AGO1蛋白的积累，且热应激诱导的AGO1可与siRNA小体（siRNA bodies）及应激颗粒（stress granules, SGs）的组分发生共定位。在热应激期间，AGO1无需依赖siRNA小体的关键组分SGS3即可高效形成凝聚体。进一步研究发现，AGO1中尚未被充分表征的、包含朊病毒样结构域（prion-like domain）的N端Poly-Q结构域（N-terminal Poly-Q domain），足以介导相分离过程。此外，仅1小时的热应激处理仅对AGO1的miRNA加载效率与靶标剪切活性产生轻度影响，提示AGO1在凝聚体中的定位更倾向于保护自身，而非在应激过程中促进基因表达重编程相关的活性。

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见解。实验整体设计：对处于对照、热应激及恢复培养条件下的拟南芥幼苗进行RNA测序（RNAseq），每组设置3次生物学重复。