1.BiFC and FACS-based CRISPR screening revealed that QKI promotes PABPN1 LLPS in colorectal cancer cells.
Mengxia LI ; Zhijie HU ; Yingye HUANG ; Yuting HAN ; Cheng LIANG ; Yuchi LIU ; Runze WU ; Xin LU ; Ke DENG ; Susu LIU ; Xin OU ; Yuwei LI ; Chao LIU ; Xuening LI ; Jingting LIANG ; Yonggui FU ; Anlong XU
Protein & Cell 2025;16(7):557-574
Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.
Humans
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Colorectal Neoplasms/genetics*
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RNA-Binding Proteins/genetics*
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Poly(A)-Binding Protein I/genetics*
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CRISPR-Cas Systems
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Flow Cytometry
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Cell Proliferation
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Cell Line, Tumor
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Cell Movement
Result Analysis
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