iProXProteomicsDahua ChenDrosophila Melanogasterhttp://www.iprox.org/page/project.html?id=IPX0004115000chendh@ioz.ac.cnInstitute of Biomedical Research, Yunnan UniversityiProXAGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. Here, we unexpectedly discover that fly and human AGO proteins, which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P₂. The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, our study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination.Gao Yajie Y, Zhu Yuanxiang Y, Wang Hailong H, Cheng Ying Y, Zhao Dongbo D, Sun Qinmiao Q, Chen Dahua DfalseLipid mediated–phase separation of AGO proteins on the ER controls nascent peptide ubiquitinationwe sought to determine whether dmAGO1 and Ltn1 share common ubiquitinated nascent peptide targets. For this purpose, we performed the UBA bead-based immunoprecipitation followed by mass spec experiments to identify the ubiquitinated nascent peptide targets of dmAGO1 and Ltn1, respectively. In this assay, we used four different samples including gfp knockdown, vcp knockdown, vcp-ago1 double knockdown and vcp-ltn1 double knockdown S2 cells.Fri Feb 18 00:00:00 GMT 2022PXD031749722735325613