VCP Machinery Mediates Autophagic Degradation of Empty Argonaute

Kobayashi, Hotaka; Shoji, Keisuke; Kiyokawa, Kaori; Negishi, Lumi; Tomari, Yukihide

doi:10.1016/j.celrep.2019.07.003

Cited by 27 publications

(24 citation statements)

References 105 publications

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“…AGO1 degradation has been reported to be controlled by selective autophagy (Kobayashi et al 2019 ; Li et al 2019 ; Michaeli et al 2019 ). The P0 viral suppressor of Polerovirus is thought to trigger autophagic AGO1 degradation (Michaeli et al 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches

Wei

Hong

et al. 2020

Bot Stud

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Background Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein that was translated from potyviral RNA. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5 ′ -fragment of the PTGS-cleaved RNA degradation product. Second, the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC - Pro of turnip mosaic virus (TuMV) ( P1/HC Tu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

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Section: Discussionmentioning

confidence: 99%

Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches

Wei

Hong

et al. 2020

Bot Stud

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“…The fact that the drug MLN4924, which blocks the activity of CULLIN-based E3 ubiquitin ligases, had a more pronounced effect in blocking AGO1 degradation highlights the importance of ubiquitylation in this process. Since both E64d and CB-5083 significantly prevented AGO1 decay, we assume that FBW2 promotes AGO1 vacuolar degradation, via a pathway involving CDC48, which could be similar to the mechanism described in flies (Kobayashi et al, 2019b). Elucidating the molecular and cellular determinants of this pathway will represent an important goal in the future.…”

Section: Mechanism Of Fbw2-mediated Ago1 Degradationmentioning

confidence: 78%

“…In animal cells, AGOs have been shown to undergo proteasomal degradation (Johnston et al, 2010) (Chinen and Lei, 2017) (Han et al, 2020) (Shi et al, 2020), but also degradation via lysosomes or even selective autophagy (Gibbings et al, 2012) (Martinez and Gregory, 2013). Interestingly, at least in Drosophila S2 cells, recent work elucidated a novel pathway in which valosin-containing protein (VCP/p97/Cdc48) directs degradation of ubiquitylated empty Ago1 through the Ufd1-Npl4 adaptor subunits (Kobayashi et al, 2019b). In Arabidopsis, our pharmacological approach revealed that the proteasome is not the main route for FBW2-mediated AGO1 decay, though a small fraction of AGO1 undergoes FBW2-dependant proteasomal degradation as supported by a slight stabilisation of AGO1 protein and the appearance of a cleavage product in the presence of Bortezomib, as well as a significant enrichment of proteasomal subunits in the FBW2 IP.…”

Section: Mechanism Of Fbw2-mediated Ago1 Degradationmentioning

confidence: 99%

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

Hacquard

Clavel

Baldrich

et al. 2021

Preprint

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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.

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“…P1/HC-Pro of TuMV speci cally primes posttranslational AGO1 degradation AGO1 degradation has been reported to be controlled by selective autophagy (Kobayashi et al, 2019;Li et al, 2019;Michaeli et al, 2019). The P0 viral suppressor of Polerovirus is thought to trigger autophagic AGO1 degradation (Michaeli et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

Wei

Hong

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5'-fragment of the PTGS-cleaved RNA degradation product. Second, specifically the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion: Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

show abstract

VCP Machinery Mediates Autophagic Degradation of Empty Argonaute

Abstract: Highlights d The empty state of Drosophila Ago1 is degraded by autophagy d Empty Ago1 is recognized by VCP, a mediator for selective autophagy d The Ufd1-Npl4 heterodimer links empty Ago1 and VCP d VCP-Ufd1-Npl4 machinery ensures proper gene silencing by microRNAs

Cited by 27 publications

References 105 publications

Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches

Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches

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

Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

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