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ERC

PHAGORISC Report Summary

Project ID: 338904
Funded under: FP7-IDEAS-ERC
Country: France

Periodic Report Summary 2 - PHAGORISC (Connecting RNA and protein degradation machineries)

This ERC program aims to provide insights into the regulatory mechanisms that control and mediate the plant Arabidopsis ARGONAUTE1 (AGO1) proteins turnover, and to identify viral and endogenous machineries involved.
The first Work Package (WP1) aims to get a deeper understanding on how the viral encoded F-box protein P0 destabilizes AGO1. In this WP, we succeeded to immunoprecipitate Arabidopsis AGO1 under normal conditions and upon induction of the viral encoded P0 protein. Series of IP-MS assays allowed us to establish the so-called AGO1 interactome in normal growth conditions and also to identify interesting proteins that specifically interact with AGO1 when P0 is induced. Among them, not surprisingly we identified RNA binding proteins and components of the translational machinery consistent with AGO1 participating in translational repression, but we also identified membrane trafficking proteins, regulatory proteins and other components that might reveal novel cellular functions for AGO1. At the molecular level, we could define the “degron” in the sequence of AGO1 that is required for P0-mediated degradation and could demonstrate that this domain is involved in small RNA duplex unwinding. Mutations in this “degron” fully stabilize AGO1 protein in presence of P0 and several mutant lines are currently tested for polerovirus resistance. We also aimed to obtain structural insights into the SCFP0 complex. Though we could produce an in vitro active SCFP0 complex, at present we failed to obtain crystals. Finally, we discovered that the SCFP0 does not induce massive ubiquitylation in plants, but specifically acts on several AGO proteins. Therefore our aim to establish the P0-dependant ubiquitylome is not anymore pertinent and Task5 has been dropped from the program.
The second Work Package (WP2) aims to characterize the proteolytic machinery mediating P0-dependant AGO1 degradation. In contrast to our predictions, we found that in autophagy mutant lines, P0-mediated AGO1 degradation could not be fully blocked, but only delayed indicating that P0 can override autophagy blockage or that P0-mediated AGO1 degradation takes a non-conventional pathway for degradation. To identify which components could be involved in this process, we undertook a forward genetic screen by EMS mutagenesis to identify suppressors of P0. The sequencing of some of these mutants is currently underway. In parallel, we made also important progresses in generating pAGO1::GFP-AGO1 reporter lines combined with different cellular markers to check for their co-localizations before and upon P0 induction. Multispectral live imaging should soon allow us to determine more precisely by which compartment(s) AGO1 is delivered to the vacuole.
The third Work Package (WP3) aims to unravel endogenous mechanisms of AGO1 turnover. For instance, in the absence of efficient miRNA biogenesis and accumulation, AGO1 protein rapidly decays. Moreover we speculated that AGO1 turnover might also be regulated during stress, when the small RNA repertoires dynamically change in response to the environment. We first planned to establish a non-degradable Arabidopsis AGO1 (ndAGO1) mutant line, which would be instrumental to determine the importance of AGO1 turnover during stress. Unfortunately our first attempts to establish such a tool failed, but we still hope to obtain it, either by mutating ubiquitylation sites in AGO1 or by identifying and mutating the “degron” recognized by FBW2, an endogenous F-box protein that has been involved in AGO1 turnover. Finally, we also addressed the question of AGO1 phosphorylation, at least in the context of viral infection. IP-MS analyses revealed a prominent, but constitutive, phosphorylation site in AGO1. Further experiments will determine whether additional phosphorylation events can be identified upon specific stresses and most importantly whether they play a role in AGO1 turnover.

Reported by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
France
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