| Autor: |
Jia, Yan, Zhao, Jianing, Yu, Tao, Zhang, Xue, Qi, Xiaozhen, Hao, Tongxin, Jin, Zeyuan, Zhao, Xiaoqing |
| Zdroj: |
Cellular & Molecular Biology Letters; 12/17/2022, Vol. 27 Issue 1, p1-17, 17p |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
Full text from SpringerLink