Heterozygous De Novo Truncating Mutation of Nucleolin in an ASD Individual Disrupts Its Nucleolar Localization

Nucleolin (NCL/C23; OMIM: 164035) is a major nucleolar protein that plays a critical role in multiple processes, including ribosome assembly and maturation, chromatin decondensation, and pre-rRNA transcription. Due to its diverse functions, nucleolin has frequently been implicated in pathological processes, including cancer and viral infection. We recently identified a de novo frameshifting indel mutation of NCL, p.Gly664Glufs*70, through whole-exome sequencing of autism spectrum disorder trios. Through the transfection of constructs encoding either a wild-type human nucleolin or a mutant nucleolin with the same C-terminal sequence predicted for the autism proband, and by using co-localization with the nucleophosmin (NPM; B23) protein, we have shown that the nucleolin mutation leads to mislocalization of the NCL protein from the nucleolus to the nucleoplasm. Moreover, a construct with a nonsense mutation at the same residue, p.Gly664*, shows a very similar effect on the location of the NCL protein, thus confirming the presence of a predicted nucleolar location signal in this region of the NCL protein. Real-time fluorescence recovery experiments show significant changes in the kinetics and mobility of mutant NCL protein in the nucleoplasm of HEK293Tcells. Several other studies also report de novo NCL mutations in ASD or neurodevelopmental disorders. The altered mislocalization and dynamics of mutant NCL (p.G664Glufs*70/p.G664*) may have relevance to the etiopathlogy of NCL-related ASD and other neurodevelopmental phenotypes.

ARF Impedes NPM/B23 Shuttling in an Mdm2-Sensitive Tumor Suppressor Pathway

ABSTRACT The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.

Evidence for the ability of nucleophosmin/B23 to bind ATP

By taking advantage of its ability to be retained by ATP-agarose, we have demonstrated that nucleophosmin/B23 is capable of binding ATP. The specificity of the binding was confirmed by the absence of significant binding to AMP-agarose and by its loss when nucleophosmin/B23 in nuclear extracts was preincubated with ATP. Preincubation of the nuclear extracts with other ribonucleotide triphosphates (GTP, CTP, UTP) did not compete for the binding of nucleophosmin/B23 to ATP-agarose. The purified recombinant nucleophosmin/B23 was also able to be retained by ATP-agarose. The Kd for binding of ATP to the purified recombinant nucleophosmin/B23, on the basis of retention on a nitrocellulose membrane, was 86.5±8.3 μM; the number of binding sites was 0.68 per nucleophosmin/B23 protein molecule. To determine the possible ATP-binding site of nucleophosmin/B23, various deletion clones including the two mutants in which the putative ATP-binding sequence had been deleted were constructed. Deletion of the portions of the molecule (residues 83-152 and 185-240) had little effect on the ATP binding. The C-terminal deleted mutant (residue 242 to the C-terminus deleted) lost most of its ability to be retained by ATP-agarose and to bind [α-32P]ATP on a nitrocellulose membrane. The results indicate that the C-terminal portion (residues 242-294) contains the essential ATP-binding site of nucleophosmin/B23.