The Arabidopsis F-box protein FBW2 degrades AGO1 to avoid spurious loading of illegitimate small RNA

Abstract

RNA silencing is a conserved mechanism in eukaryotes and is involved in development, heterochromatin maintenance and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA (miRNA) and small interfering RNA (siRNA)-directed silencing and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 targets proteolysis of AGO1 by a CDC48-mediated mechanism. We found that FBW2 assembles an SCF complex that recognizes the MID-PIWI domain of AGO1 and requires its C-terminal domain containing a GW motif for AGO1 turnover. We showed that FBW2 prefers the unloaded and some mutated forms of AGO1 protein. While FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA silencing activity. Interestingly, under conditions in which small RNA production or accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Hence, the non-degradable AGO1 protein assembles high molecular weight complexes and binds illegitimate small RNA leading to the cleavage of new target genes that belong to stress responses and cellular metabolic processes. Therefore, the control of AGO1 homeostasis by ubiquitin ligases plays an important role in quality control to avoid off-target cleavage.

Fraternity of old‐timers: How ubiquitin regulates miRNA functions

“…In this review, we have examined data on how the protein ubiquiti- is possible that the proteins required to degrade unloaded Ago may influence the activity of the miRNA system in some other way because there are indications that although plant FWB2 prefers to bind to unloaded and mutant AGO1, a small fraction of it binds to loaded AGO1 too. [43,121] Finally, it is clear that unloaded Ago degradation can be enforced or attenuated in response to external stimuli. [46,106] In this regard, it would be interesting to find out how widespread such regulatory destruction of unloaded Ago is and what its biological functions might be.…”

“…The selective autophagy of only the unloaded Ago is possible because its conformational structure differs from that of the loaded one. In this way, the RING-containing ubiquitin ligase Iruka recognizes lysine K514 on the surface of unloaded fly dAgo1, [42] while in plants AGO1 is ubiquitinated by the F-box containing FBW2 protein, [43] recognizing the MID-PIWI domain. The autophagy lunches once ubiquitinated Ago binds to the substrate receptor complex Ufd1-Npl4 and the mediator protein VCP in Drosophila, [44] substrate receptor NDP52 in mammals, [45] and CDC48, a distant homolog of VCP, in plants [43] (Figure 1).…”

“…In this way, the RING-containing ubiquitin ligase Iruka recognizes lysine K514 on the surface of unloaded fly dAgo1, [42] while in plants AGO1 is ubiquitinated by the F-box containing FBW2 protein, [43] recognizing the MID-PIWI domain. The autophagy lunches once ubiquitinated Ago binds to the substrate receptor complex Ufd1-Npl4 and the mediator protein VCP in Drosophila, [44] substrate receptor NDP52 in mammals, [45] and CDC48, a distant homolog of VCP, in plants [43] (Figure 1). The excess of unloaded human hAgo2 is degraded not only in lysosomes but also by proteasomes, [41,46] but the mechanism of this is unclear.…”

“…In plants, the accumulated and undisrupted AGO1 begins to bind to non-targeted short RNAs, including fragments of transposon transcripts and rRNAs. [43] Loaded with those RNA species, AGO1 directs cleavage of many off-target transcripts, whose protein products are participants in the metabolic and cellular response to stress, resulting in an overall reduction of Arabidopsis viability. Thus, the ubiquitination system not only maintains a minimally sufficient level of Ago but also ensures that the miRNA system works correctly by controlling the fraction of functionally active Ago.…”

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

“…The p35S:mRFP-AGO1 construct was obtained by Gateway LR recombination (Invitrogen) using pENTRY(ZEO)-AGO1(CDS) (Hacquard et al, 2022) and the binary vector pB7WGR2 (Karimi et al, 2005); https://gatewayvectors.vib.be/collection. This construct expresses the mRFP-AGO1(CDS) fusion protein under the regulation of the 35S promoter.…”