Article

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

Program in Cell Dynamics, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Molecular biology of the cell (Impact Factor: 5.98). 08/2008; 19(7):2870-5. DOI: 10.1091/mbc.E08-02-0128
Source: PubMed

ABSTRACT Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.

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    • "GTP bound to the central region of NS is thought to stabilize interactions between its amino-terminal basic domain and other nucleolar components, whereas NS redistributes to the nucleoplasm when GTP binding is impaired by mutation (Tsai and McKay, 2005). Coimmunoprecipitation, yeast twohybrid , and bimolecular fluorescence complementation assays demonstrated an interaction between NS and nucleophosmin (B23) (Ma and Pederson, 2008a), a multifunctional chaperone that probably participates in the later stages of ribosome assembly within the granular component of nucleoli . Similar to NS, nucleophosmin probably plays a role in cell proliferation (Szebeni et al., 2003; Grisendi et al., 2006). "
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    ABSTRACT: Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.
    Molecular biology of the cell 09/2009; 20(20):4424-34. DOI:10.1091/mbc.E08-06-0592 · 5.98 Impact Factor
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    • "Finally, miRNAs may concentrate in the nucleolus because they are active as regulatory modification/processing components themselves. For example, small noncoding RNAs have been implicated in the control of ribosomal DNA transcription (Mayer et al. 2008), and snoRNAs guide modification of rRNA and some snRNAs in the nucleolus (Boisvert et al. 2007; Matera et al. 2007), as well as perhaps modulating the activity of ADAR2 (Vitali et al. 2005). However, neither miR-206 nor the nucleolus-enriched miRNAs identified in the present study were localized at the rDNA transcription sites (fibrillar centers) or to the DFC, where most snoRNA-guided rRNA modifications are thought to occur, but rather were clustered in the GC, a region that is known to be the site of late stages of rRNA processing. "
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    ABSTRACT: There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have recently been ascribed to the granular component of the nucleolus.
    RNA 08/2009; 15(9):1705-15. DOI:10.1261/rna.1470409 · 4.62 Impact Factor
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    • "Among the three nucleostemin-binding proteins in the nucleolus, ARF and B23 have been known to participate in both the cell cycle regulatory and ribosomal-processing pathways. The other protein, RSL1D1, has recently been shown to delay replicative senescence in human fi broblast cells by regulating PTEN ( Ma et al., 2008 ). Could RSL1D1 be the primary nucleolar target of nucleostemin that confers its anti-senescence activity, as seen in mouse embryonic fi broblast cells ( Zhu et al., 2006 ); or could "
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    ABSTRACT: The life of the nucleolus has proven to be more colorful and multifaceted than had been envisioned a decade ago. A large number of proteins found in this subnuclear compartment have no identifiable tie either to the ribosome biosynthetic pathway or to the other newly established activities occurring within the nucleolus. The questions of how and why these proteins end up in this subnuclear compartment remain unanswered and are the focus of intense current interest. This review discusses our thoughts on the discovery of nonribosomal proteins in the nucleolus.
    The Journal of Cell Biology 04/2009; 184(6):771-6. DOI:10.1083/jcb.200812014 · 9.69 Impact Factor
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