Nucleophosmin acute myeloid leukemia mutant is unable to bind G-quadruplex sequences at ribosomal DNA in vitro.

Nucleophosmin (NPM1) is an abundant phospho-protein that plays a key role in ribosome biogenesis. NPM1 binds nucleic acids and has intrinsic RNAase and chaperone activities. Although mainly localized at nucleoli, NPM1 continuously shuttles between the nucleus and the cytoplasm to fullfill its functions. The gene encoding NPM1 is mutated in 50–60% of normal kariotype acute myeloid leukemia (AML) patients. These mutants are stably localized in the cytoplasm of leukemic cells due to the critical changes at the NPM1 C-terminus domain: i) loss of tryptophan residues 288 and 290 (or 290 alone), which destabilizes the C-terminal domain; ii) generation of a new Nuclear Export Signal (NES) motif. Both alterations are critical for perturbing NPM1 mutant traffic and for the underlying aberrant accumulation of NPM1 in the cytoplasm of leukemic cells. We investigated the structural determinants of NPM1 nucleolar localization in vitro by means of Surface Plasmon Resonance (SPR). We showed that a domain encompassing the last 70 residues of the protein (NPM1-C70) binds with high affinity oligonucleotide sequences with G-quadruplex structure found at the non-coding strand of ribosomal DNA. Importantly, the most common leukemic NPM1 variant C-terminal domain, loses this DNA binding activity being completely unfolded. Since mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex binding activity, we suggest that NPM1 nucleolar localization depends on its interaction with G-quadruplex regions at rDNA, which is impaired by AML-associated mutations.