BiFC and FACS-based CRISPR screening revealed that QKI promotes PABPN1 LLPS in colorectal cancer cells

Protein Cell. 2025 Jul 19;16(7):557-574. doi: 10.1093/procel/pwaf022.

Abstract

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.

Keywords: CRISPR/Cas9 screening; PABPN1; QKI; colorectal cancer; liquid–liquid phase separation.

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • Colorectal Neoplasms* / genetics
  • Colorectal Neoplasms* / metabolism
  • Colorectal Neoplasms* / pathology
  • Flow Cytometry
  • Humans
  • Poly(A)-Binding Protein I* / genetics
  • Poly(A)-Binding Protein I* / metabolism
  • RNA-Binding Proteins* / genetics
  • RNA-Binding Proteins* / metabolism

Substances

  • RNA-Binding Proteins
  • Poly(A)-Binding Protein I
  • QKI protein, human
  • PABPN1 protein, human