The Regulation Of Argonaute2 Ubiquitination

Objective:RNAi(RNA interference) is a biological process that regulating gene silencing or gene inhibition in eukaryocyte. RNAi is mediated by small non-coding RNA which primarily include microRNAs(miRNAs) and short interfering RNAs(siRNAs). siRNA or miRNA can be recognized by Argonaute2(Ago2) protein to form the RNAi effector known as the RNA-induced silencing complex(RISC). Then siRNA or miRNA guide the RISC to recognize and cleave the target mRNA. Since RISC plays a key role in RNAi process, its core component Ago2 is considered as the catalytic engine in the RNAi apparatus. It can help us to understand the RNAi process by studying the regulation of Ago2. We screened Ago2 interaction protein and determined that PSMC3 can stabilize Ago2 and thus raise the RNAi efficiency in our previous work. But the molecular mechanism remains unclear. Here we aim to reveal that PSMC3 can facilitate USP14 to de-ubiquitiates Ago2 and inhibite its degradation in 26 S proteasome, thus stabilize Ago2. And explore the role of RBBP6 on Ago2 ubiquitination. Methods:First, both lysosome inhibitor and proteasome inhibitor were used to determin where Ago2 protein was degraded. And Ago2 mutations and immunoprecipitation assay were used to map the ubiquitination site of Ago2. After that, coimmuneprecipitation and immunofluorescence technique were employed to study the interaction between USP14 and Ago2. Cycloheximide, Western blot and immunoprecipitation assays were used to verify that if USP14 can de-ubiquitiante Ago2 and hence to regulate its protein level. In addition, USP14 mutations and coimmuneprecipitation assay were used to map USP14, PSMC3 and Ago2 interaction domains. Western blot assay was used to determin the involvement of PSMC3 in the regulation of Ago2 by USP14. Besides, co-immuneprecipitation assay was used to verify the interaction between RBBP6 and Ago2. Immuneprecipitation and Western blot assays were use to explore the role of RBBP6 on Ago2 protein. At last, Western blot assay was use to determin the effection of RBBP6, PSMC3 and USP14 on RNAi efficiency. Result:Proteasome inhibitor promotes the accumulation of ubiquitinated Ago2. USP14 interact with both PSMC3 and Ago2 and co-localized with them. Depletion of USP14 leads to a shorter half-life of Ago2 protein. USP14 inhibitor promotes the accumulation of ubiquitinated Ago2 in cells. USP14 regulates Ago2 protein level and MG132 treatment counteracts this effection. RBBP6 interacts with Ago2 and control the Ago2 protein level. MG132 treatment attenuates this effection. The alteration of RBBP6, PSMC3 and USP14 expression level affect RNAi efficiency. Conclusion:We reveal the process of Ago2 ubiquitination and de-ubiquitination. USP14 is identified as deubiquitinating enzymes of Ago2. USP14 regulates the deubiquitination of Ago2 and prevent its subsequently degradation in 26 S proteasome. PSMC3 is proved to facilitate USP14 to de-uibiquitinate Ago2. Additonally, RBBP6 is suggested to promote Ago2 ubiquitination.