APE1 condensation in nucleoli of non-cancer cells depends on rRNA transcription and forming G-quadruplex RNA structures

Abstract

APE1 [apurinic/apyrimidinic (AP) endodeoxyribonuclease 1] is the main endonuclease of the base excision repair pathway acting on abasic (AP) sites in DNA. APE1 is an abundant nuclear protein, and improper expression or localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 subcellular distribution and expression are associated with cancer development, suggesting the importance of a fine-tuning mechanism for APE1 activities. Recent works highlighted the presence of APE1 within nucleoli of cancer cells and the ability of APE1 to form biomolecular condensate. However, whether secondary structures of ribosomal RNA (rRNA) influence the nucleolar localization of APE1 remains poorly understood. Since protein overexpression can result in artificial nucleolar accumulation, it is imperative to have appropriate cellular models to study APE1 trafficking under physiological conditions. To address this issue, we generated a murine embryonic stem cell line expressing endogenous fluorescent-tagged APE1. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription and is modulated by different genotoxicants. In vitro experiments showed that APE1 condensate formation depends on RNA-forming G-quadruplex structures and relies on critical lysine residues. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and the formation of RNA-dependent APE1 nucleolar condensates.