Dynamic phosphorylation of TCOF1 controlled by CK2 and PPM1D regulates nucleolar condensation and RNA polymerase I activity

Abstract

The nucleolus is a membrane-less organelle essential for ribosomal DNA (rDNA) transcription and ribosome biogenesis, yet the mechanisms regulating its dynamic assembly remain incompletely understood. Here, we identify the phosphatase protein phosphatase magnesium-dependent 1 delta (PPM1D) as a key regulator of the nucleolar architecture via dephosphorylation of the scaffold protein TCOF1. By combining super-resolution microscopy and domain mapping, we show that PPM1D localizes to the nucleolus through a conserved motif at the C-terminus. Phosphoproteomic analysis and structural modeling revealed that PPM1D targets casein kinase 2 (CK2)-phosphorylated sites within the low-complexity repeat domain of TCOF1, which impacts its interaction with RNA polymerase I (Pol I), immobilization in the fibrillar centres, and nucleolar condensation. Disruption of the phosphorylation balance by loss of PPM1D or inhibition of CK2 leads to nucleolar disorganization and reduced rDNA transcription. Finally, we demonstrate that pharmacological inhibition of PPM1D sensitizes cells to RNA Pol I inhibitors, in both p53-dependent and -independent contexts. Together, we uncover a reversible phosphorylation switch that governs TCOF1 dynamics and its nucleolar function, with potential implications for targeting ribosome biogenesis in cancer.