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PSMC3 promotes RNAi by maintaining AGO2 stability through USP14

BACKGROUND: Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting...

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Autores principales: Jia, Yan, Zhao, Jianing, Yu, Tao, Zhang, Xue, Qi, Xiaozhen, Hao, Tongxin, Jin, Zeyuan, Zhao, Xiaoqing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9759854/
https://www.ncbi.nlm.nih.gov/pubmed/36528617
http://dx.doi.org/10.1186/s11658-022-00411-y
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author Jia, Yan
Zhao, Jianing
Yu, Tao
Zhang, Xue
Qi, Xiaozhen
Hao, Tongxin
Jin, Zeyuan
Zhao, Xiaoqing
author_facet Jia, Yan
Zhao, Jianing
Yu, Tao
Zhang, Xue
Qi, Xiaozhen
Hao, Tongxin
Jin, Zeyuan
Zhao, Xiaoqing
author_sort Jia, Yan
collection PubMed
description BACKGROUND: Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments. METHODS: The yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed. RESULTS: We identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi. CONCLUSION: Our findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s11658-022-00411-y.
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spelling pubmed-97598542022-12-19 PSMC3 promotes RNAi by maintaining AGO2 stability through USP14 Jia, Yan Zhao, Jianing Yu, Tao Zhang, Xue Qi, Xiaozhen Hao, Tongxin Jin, Zeyuan Zhao, Xiaoqing Cell Mol Biol Lett Research BACKGROUND: Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments. METHODS: The yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed. RESULTS: We identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi. CONCLUSION: Our findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s11658-022-00411-y. BioMed Central 2022-12-17 /pmc/articles/PMC9759854/ /pubmed/36528617 http://dx.doi.org/10.1186/s11658-022-00411-y Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research
Jia, Yan
Zhao, Jianing
Yu, Tao
Zhang, Xue
Qi, Xiaozhen
Hao, Tongxin
Jin, Zeyuan
Zhao, Xiaoqing
PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title_full PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title_fullStr PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title_full_unstemmed PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title_short PSMC3 promotes RNAi by maintaining AGO2 stability through USP14
title_sort psmc3 promotes rnai by maintaining ago2 stability through usp14
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9759854/
https://www.ncbi.nlm.nih.gov/pubmed/36528617
http://dx.doi.org/10.1186/s11658-022-00411-y
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