Final Report Summary - PHAGORISC (Connecting RNA and protein degradation machineries)
RNA silencing involves processing of dsRNA by the enzyme Dicer, into small RNAs, 21-to-25 nucleotides in length. One of the two RNA strands is then incorporated into a protein complex called RISC (RNA induced silencing complex) that invariably contains a member of the conserved ARGONAUTE protein family. RNA silencing is key for the regulation of development in animals and plants, but plays also a major antiviral role in plants and invertebrates. This ERC program aimed to provide insights into the regulatory mechanisms that control plant Arabidopsis ARGONAUTE1 (AGO1) protein homeostasis and to identify viral and endogenous machineries involved in its turnover. To answer to these questions that received so far little attention, we undertook a multidisciplinary approach, combining molecular and cell biology, genetics, biochemistry and structural biology.
Work Package (WP1) provided insights into the mode of action of the Viral Suppressors of RNA silencing (VSRs) P0. It was previously shown that the polerovirus P0 protein hijacks the host SCF-type ubiquitin E3 ligase machinery to destabilize AGO1 in order to impair host anti-viral defence. In this WP, we succeeded to establish the so-called Arabidopsis AGO1 protein interactome in normal growth conditions and also to identify proteins that specifically interact with AGO1 when P0 is expressed. Among them, not surprisingly we identified components of the translational machinery consistent with AGO1 participating in translational repression, but we also identified uncharacterized RNA binding proteins, regulatory proteins and proteins involved in nucleo-cytoplasmic and endocytic trafficking, which are still currently under investigation. At the molecular level, we could define the “degron”, the minimal element within AGO1 that is sufficient for its recognition and degradation by the SCFP0 ubiquitin ligase. Mutations in this “degron” fully stabilize AGO1 protein in presence of P0 and we also observed that the expression of the non-degradable AGO1 confers at least a partial resistance to the phloem-restricted Turnip mosaic virus (TuMV) in Arabidopsis. We took advantage of this mutation, which is located in the still poorly characterized AGO1 DUF1785, to study the function of this protein domain. Thus, we found that the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing.
Work Package (WP2) revealed novel information on the proteolytic machinery mediating P0-dependant AGO1 degradation and its trafficking to the vacuole. In particular, we found that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8- Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing activity. In parallel, a forward genetic screen by EMS mutagenesis was conducted to identify suppressors of P0, revealing some additional candidate genes, which are involved in this degradation pathway.
Work Package (WP3) aimed to unravel endogenous mechanisms of AGO1 proteostasis that occur in a non-viral context. For instance, we have previously shown that in the absence of efficient miRNA biogenesis and accumulation, AGO1 protein rapidly decays. Here, we also identified several stress conditions, which affected AGO1 protein turnover. The most significant progress made in WP3 concerned the functional characterization of an endogenous F-box protein called FBW2. We elucidated the mechanism by how this F-box recognizes AGO1 and unraveled some of its physiological functions. Finally, we investigated AGO1 post-translational modifications and identified several novel phosphorylation sites.
Work Package (WP1) provided insights into the mode of action of the Viral Suppressors of RNA silencing (VSRs) P0. It was previously shown that the polerovirus P0 protein hijacks the host SCF-type ubiquitin E3 ligase machinery to destabilize AGO1 in order to impair host anti-viral defence. In this WP, we succeeded to establish the so-called Arabidopsis AGO1 protein interactome in normal growth conditions and also to identify proteins that specifically interact with AGO1 when P0 is expressed. Among them, not surprisingly we identified components of the translational machinery consistent with AGO1 participating in translational repression, but we also identified uncharacterized RNA binding proteins, regulatory proteins and proteins involved in nucleo-cytoplasmic and endocytic trafficking, which are still currently under investigation. At the molecular level, we could define the “degron”, the minimal element within AGO1 that is sufficient for its recognition and degradation by the SCFP0 ubiquitin ligase. Mutations in this “degron” fully stabilize AGO1 protein in presence of P0 and we also observed that the expression of the non-degradable AGO1 confers at least a partial resistance to the phloem-restricted Turnip mosaic virus (TuMV) in Arabidopsis. We took advantage of this mutation, which is located in the still poorly characterized AGO1 DUF1785, to study the function of this protein domain. Thus, we found that the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing.
Work Package (WP2) revealed novel information on the proteolytic machinery mediating P0-dependant AGO1 degradation and its trafficking to the vacuole. In particular, we found that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8- Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing activity. In parallel, a forward genetic screen by EMS mutagenesis was conducted to identify suppressors of P0, revealing some additional candidate genes, which are involved in this degradation pathway.
Work Package (WP3) aimed to unravel endogenous mechanisms of AGO1 proteostasis that occur in a non-viral context. For instance, we have previously shown that in the absence of efficient miRNA biogenesis and accumulation, AGO1 protein rapidly decays. Here, we also identified several stress conditions, which affected AGO1 protein turnover. The most significant progress made in WP3 concerned the functional characterization of an endogenous F-box protein called FBW2. We elucidated the mechanism by how this F-box recognizes AGO1 and unraveled some of its physiological functions. Finally, we investigated AGO1 post-translational modifications and identified several novel phosphorylation sites.