Gene

Cytoplasm

Cell biology

Small nuclear ribonucleoprotein

Biology

Phosphoproteomics

Protein phosphorylation

Biochemistry

Protein kinase A

Phosphorylation

Stable isotope labeling by amino acids in cell culture

Computational biology

Spliceosome

Nucleolus

Ribonucleoprotein

RNA splicing

Chemistry

Proteomics

SR protein

Phosphopeptide

Proteome

snRNP

Nature Chemical Biology

Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein-RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.

Systematic discovery of biomolecular condensate-specific protein phosphorylation