PSMC3 promotes RNAi by maintaining AGO2 stability through USP14.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Argonaute 2 (AGO2) is a key protein in RNA interference (RNAi), essential for gene silencing.
• Understanding AGO2 interactions is crucial for deciphering RNAi pathway regulation.

Purpose of the Study:

• To identify novel AGO2-interacting proteins using yeast two-hybrid screening.
• To elucidate the mechanism by which interacting proteins regulate AGO2 stability and RNAi function.

Main Methods:

• Yeast two-hybrid screening to identify AGO2 binding partners.
• Co-immunoprecipitation, immunofluorescence, and western blotting to confirm interactions and analyze protein levels.
• EGFP fluorescence assays and RT-qPCR to assess RNAi activity and mRNA levels.
• Ubiquitination and deubiquitination assays to investigate protein turnover.

Main Results:

• PSMC3 (proteasome 26S subunit, ATPase, 3) was identified as a novel AGO2 binding partner.
• PSMC3 binds AGO2 independently of RNA, requiring its N-terminal coiled-coil motif.
• PSMC3 depletion impairs small RNA-mediated cleavage and reduces AGO2 protein levels by increasing its ubiquitination and proteasomal degradation.
• PSMC3 facilitates USP14-mediated deubiquitination of AGO2, thereby stabilizing the protein and promoting RNAi.

Conclusions:

• PSMC3 is essential for maintaining AGO2 stability through deubiquitination.
• PSMC3 plays a critical role in regulating RNA interference efficiency by stabilizing AGO2.