PhD-2025-18: Mechanism and role of Arabidopsis AGO1 phase separation

Team leader: Pascal GENSCHIK

PhD supervisor: Pascal GENSCHIK

In the model plant Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 is also an important regulator of antiviral defense, as its mutation enhances susceptibility to different RNA viruses. Under normal conditions, AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. It has recently been shown that under heat stress AGO1 protein accumulates in cytosolic condensates where it colocalizes with components of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation. These recent results raise a number of important questions. Do other abiotic or even biotic stresses have similar effects on AGO1 subcellular localization. Do AGO1 condensates obtained under different stresses have a similar (protein and RNA) composition? Will the deletion of the entire N-terminal Poly-Q domain of AGO1 be sufficient to block phase separation? Does AGO1 localization in condensates requires post-translational modifications? Given the importance of post-transcriptional gene silencing in plant development, stress responses, and antiviral defense, it will also be essential to determine to which extent the ability of AGO1 to phase separate will affect these different functions. The PhD work will employ different approaches and methods to answer these questions. These include the generation of AGO1 mutations impairing its phase separation in vivo, identifying AGO1 interactors by proximity labeling (TurboID) coupled with mass spectrometry, performing transcriptome and small RNA sequencing to unravel the physiological and molecular functions of AGO1 under stress.

Keywords: RNA silencing, ARGONAUTE1, phase separation, stress responses, post-translational modifications, Arabidopsis

– Michaeli S, Clavel M, Lechner E, Viotti C, Wu J, Dubois M, Hacquard T, Derrien B, Izquierdo E, Lecorbeiller M, Bouteiller N, De Cilia J, Ziegler-Graff V, Vaucheret H, Galili G and Genschik P (2019) The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1. Proc Natl Acad Sci USA 116: 22872-22883.

– Hacquard T, Clavel M, Baldrich P, Lechner E, Pérez-Salamó I, Schepetilnikov M, Derrien B, Dubois M, Hammann P, Kuhn L, Brun D, Bouteiller N, Baumberger N, Vaucheret H, Meyers B and Genschik (2022) The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to avoid spurious loading of illegitimate small RNA. Cell Rep. 39: 110671.

– Blagojevic A, Baldrich P, Schiaffini M, Lechner E, Baumberger N, Hammann P, Elmayan T, Garcia D, Vaucheret H, Meyers BC, Genschik P (2024) Heat stress promotes Arabidopsis AGO1 phase separation and association with stress granule components. iScience 27(3):109151.