Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Abstract

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

“…29 Consistently with this picture, it was also shown that treatment of OCI-AML2 cells, bearing wild-type NPM1 at both alleles, with the G-quadruplex ligand TmPyP4 completely displaced NPM1 from nucleoli to the nucleoplasm. 34 Collectively, these data showed that a direct correlation exists between NPM1 C-terminal domain folding, G-quadruplex binding, and the nucleolar localization of the protein. Furthermore, as G-quadruplex regions are found at the nontemplate strand of the rDNA gene, 58 they will also be present in the pre-rRNA precursor, where they may be recognized by NPM1 C-terminal domain during prerRNA processing, even though this is still to be confirmed experimentally.…”

“…Conversely, basespecific interactions and interactions with nucleotides belonging to the G-quadruplex connecting loops appear marginal, explaining why NPM1 recognizes with similar affinities several G-quadruplexes with varying loop structures. 34,45 As mentioned above, a lysine-rich region that flanks the terminal three-helix bundle (residues 225-242) proved necessary for high-affinity recognition of all oligonucleotides tested, despite their structure. 45 The overall positive charge of this tail seems to be important for recognition, as shown by the loss of G-quadruplex affinity displayed by a double lysine to alanine mutant (K229A-K230A) in the tail.…”

“…58 Consistently with its role as a Gquadruplex-binding protein, NPM1 was later shown to bind these regions both in vitro and in vivo. 34 This activity was completely lost by the unstructured leukemic mutant A C-terminal domain. In addition, a refolding mutant A variant, where both tryptophans were reinserted in their topological position in the context of the mutant A sequence, reacquired (i) a folding similar to the wild-type domain, (ii) G-quadruplex binding at rDNA regions, 34 and (iii) nucleolar localization.…”

“…34 This activity was completely lost by the unstructured leukemic mutant A C-terminal domain. In addition, a refolding mutant A variant, where both tryptophans were reinserted in their topological position in the context of the mutant A sequence, reacquired (i) a folding similar to the wild-type domain, (ii) G-quadruplex binding at rDNA regions, 34 and (iii) nucleolar localization. 29 Consistently with this picture, it was also shown that treatment of OCI-AML2 cells, bearing wild-type NPM1 at both alleles, with the G-quadruplex ligand TmPyP4 completely displaced NPM1 from nucleoli to the nucleoplasm.…”

“…29 Folding analysis of the C-terminal domain of such variant protein demonstrated that it folds similarly to the wild type. 34 Therefore, it appears that AML-associated NPM1 mutations drive the cytoplasmic localization of the protein by combining two different effects. 35 First, rather than identifying a sequence-specific NoLS for NPM1, we should consider that a properly folded C-terminal domain as a whole is the NoLS.…”