Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

Abstract

Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo-or hetero-oligomers through its N-Terminal region, and bind DNA and RNA through its C-Terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-Associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.