Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State

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

doi:10.1016/j.molcel.2018.10.033

Cited by 37 publications

(52 citation statements)

References 75 publications

(82 reference statements)

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“…( C ) In vitro loading reactions were done using lysates prepared from S2 cells transiently expressing the indicated wild-type or F2V2 mutant AGO1 tagged with FLAG at the N-terminus (46,61). For F2V2 mutant, the level of AGO1 protein was lower in the cell extract potentially due to the mechanism degrading empty AGO1 (Supplementary Figure S10A) (68,69). The effects appear to be post-transcriptional because both plasmids produced similar levels of AGO1 transcript (Supplementary Figure S10B).…”

Section: Resultsmentioning

confidence: 99%

Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

Goh

Okamura

2018

Nucleic Acids Research

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Argonaute proteins play important roles in gene regulation with small RNAs (sRNAs) serving as guides to targets. Argonautes are believed to bind sRNAs in a sequence non-specific manner. However, we recently discovered that Argonautes selectively load endogenous single-stranded (ss) RNAs, suggesting that Argonaute loading may conform to sequence specificity. To identify sequences preferred for Argonaute loading, we have developed HIgh-throughput Sequencing mediated Specificity Analysis (HISSA). HISSA allows massively parallel analysis of RNA binding efficiency by using randomized oligos in in vitro binding assays and quantifying RNAs by deep-sequencing. We chose Drosophila as a model system to take advantage of the presence of two biochemically distinct Argonautes, AGO1 and AGO2. Our results revealed AGO2 loading to be strongly favored by G-rich sequences. In contrast, AGO1 showed an enrichment of the ‘GAC’ motif in loaded species. Reanalysis of published sRNA sequencing data from fly tissues detected enrichment of the GAC motif in ssRNA-derived small RNAs in the immunopurified AGO1-complex under certain conditions, suggesting that the sequence preference of AGO1-loading may influence the repertoire of AGO1-bound endogenous sRNAs. Finally, we showed that human Ago2 also exhibited selectivity in loading ssRNAs in cell lysates. These findings may have implications for therapeutic ssRNA-mediated gene silencing.

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Section: Resultsmentioning

confidence: 99%

Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

Goh

Okamura

2018

Nucleic Acids Research

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“…SDN1/2 is involved in this process, as the sdn1 sdn2 double knock-out mutant significantly suppresses developmental defects caused by STTM (Yan et al, 2012). It is probable that TM may lead to conformational and/or post-translational modification changes in AGO1 (Golden et al, 2017; Huberdeau et al, 2017; Kobayashi et al, 2019), which releases the 3′ end of miRNAs from the PAZ domain of AGO1 and increases their susceptibility to SDN1/2 (Chen et al, 2018). Moreover, loss of function in an F-box family protein HWS (Hawaiian Skirt) suppresses both MIM and STTM-induced developmental defects (Lang et al, 2018; Mei et al, 2018).…”

Section: Target Rnas Affecting Mirna Stabilitymentioning

confidence: 99%

Plant microRNAs: Biogenesis, Homeostasis, and Degradation

2019

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MicroRNAs (miRNAs), a class of endogenous, tiny, non-coding RNAs, are master regulators of gene expression among most eukaryotes. Intracellular miRNA abundance is regulated under multiple levels of control including transcription, processing, RNA modification, RNA-induced silencing complex (RISC) assembly, miRNA-target interaction, and turnover. In this review, we summarize our current understanding of the molecular components and mechanisms that influence miRNA biogenesis, homeostasis, and degradation in plants. We also make comparisons with findings from other organisms where necessary.

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“…In the fission yeast Schizosaccharomyces pombe, it was hypothesised that the accumulation of unloaded Ago1, which is involved in transcriptional silencing, might be problematic to cells as it could generate inactive complexes poisoning the activity of small RNA-programmed RNA-induced transcriptional silencing (RITS) complexes (Holoch and Moazed, 2015). As discussed above, in Drosophila, the ubiquitin E3 ligase Iruka eliminates the empty form of Ago1 (Kobayashi et al, 2019a). In this work, the authors suggested that this mechanism would be particularly relevant for dysfunctional forms of Ago1, potentially originated from translational errors or incorrect folding and locked in an empty state.…”

Section: Why Is It Important To Degrade Ago1?mentioning

confidence: 99%

“…Nevertheless, for both proteasomal and autophagy-dependent degradation pathways of AGO proteins, the identity of the ubiquitin E3 ligase(s) involved remained unclear until recently. A first hint came with the identification of a RING-type E3 ubiquitin ligase from Drosophila named Iruka, which preferentially binds and ubiquitylates empty Ago1 (Kobayashi et al, 2019a). Moreover, in mammals, it was recently shown that extensive miRNA-target complementarity can trigger AGO proteasomal decay exposing miRNA for degradation (Shi et al, 2020) (Han et al, 2020).…”

Section: Introductionmentioning

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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Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State

Cited by 37 publications

References 75 publications

Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

Plant microRNAs: Biogenesis, Homeostasis, and Degradation

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

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