Protein NPM3 Interacts with the Multifunctional Nucleolar Protein B23/Nucleophosmin and Inhibits Ribosome Biogenesis

Abstract

Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication, and regulation of p53 expression. A yeast two-hybrid screen was performed in a search for interaction partners of B23. The complementary DNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones. Protein NPM3 and its interaction with B23 were further characterized. Endogenous B23 was able to be co-immunoprecipitated with NPM3, and this complex was resistant to ribonuclease treatment and high concentrations of salt. The N-terminal 35-90 amino acids of B23 were found to be required for their interaction. Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes, one containing B23 and 28 S ribosomal RNA (rRNA) and another composed of B23, NPM3, and other proteins, but no RNA. NPM3 was localized in the nucleolus, and its nucleolar localization depended on active rRNA transcription. In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing. Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.Ribosome biogenesis in eukaryotic cells is a multistep process that takes place primarily in the nucleolus where the individual stages of assembly correlate with specific subclasses of ultrastructures (1-4). The process begins with transcription of the ribosomal DNA at the border between the fibrillar center and the dense fibrillar components of the nucleolus. The product of transcription, 47 S pre-ribosomal RNA (pre-rRNA) 1 in mammals, is processed into smaller pre-rRNA intermediates, which finally become 28, 5.8, and 18 S rRNA. The nascent pre-ribosomal particles of the dense fibrillar components eventually mature into granular components, with ribosomal proteins added at various steps in the process. Numerous nonribosomal proteins and small nucleolar RNA participate in these steps.Protein B23 (NPM1, nucleophosmin) is an abundant nucleolar non-ribosomal protein whose locations and multiple activities suggest it plays a role in ribosome biogenesis. This protein is primarily localized to the granular component region with lesser amounts in the dense fibrillar components of the nucleolus (5-8). The nucleolar localization of B23 is dependent on the presence of active rDNA transcription (9), and it is found in association with maturing pre-ribosomal RNP particles (10, 11). In vitro experiments indicate B23 has nucleic acid binding activity and ribonuclease activities (12-17). The nucleic acid binding activity has been mapped to its C-terminal end (17,18) and is believed to be important in its nucleolar localization (15).In support of this, a splicing variant, B23.2, in which the C-terminal 35-amino acid sequence is absent, exists both in the nucleoplasm and cytoplasm (15,17). More recent studies provide evidence for a direct role ...

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…The oligomerization of NPM1 is essential for at least some of its functions in vivo, likely through allowing simultaneous and closely juxtaposed binding of multiple targets or of multi-subunit complexes (Thyagarajan et al 1998;Xia et al 2013). Interestingly, NPM3 has not been reported to form homopentameric rings and is unable to act as a histone chaperone in its own right; instead, it assembles into hetero-oligomers with NPM1, in doing so promoting the histone chaperone activity of NPM1 while repressing its RNA-binding and ribosome biogenesis activities (Finn et al 2012;Huang et al 2005;Okuwaki et al 2012). D r a f t 8 Consistent with its identification as a nucleolar phosphoprotein, NPM1 undergoes extensive posttranslational modifications including phosphorylation, ubiquitination and SUMOylation, arginine methylation, lysine acetylation and others (Colombo et al 2011), demonstrating it to be a highly regulated protein in vivo.…”

Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters

“…Overexpression and tumorigenic activation of the Smoothened (SMO) protooncogene mediates c-myc overexpression, suggesting that SMO may also be a prognostic factor in HCC tumorigenesis (Sicklick et al, 2006). Nucleophosmin (NPM) is a major nucleolar phosphoprotein implicated in multiple cellular functions, including ribosomal protein assembly and transport (Verheggen et al, 2000;Huang et al, 2005), centrosome duplication (Okuda et al, 2000;Okuda, 2002;Grisendi et al, 2005), molecular chaperone activity in preventing protein aggregation (Hingorani et al, 2000;Szebeni et al, 2003), and regulating the activity of the tumour suppressors p53 (Colombo et al, 2002;Li et al, 2004;Maiguel et al, 2004) and p14 ARF (Itahana et al, 2003;Bertwistle et al, 2004;Brady et al, 2004). Earlier studies have shown that the level of NPM is markedly and promptly increased in association with cell commitment to mitogenesis (Feuerstein and Mond, 1987;Feuerstein et al, 1988).…”

Long-Term Evolution and Functional Diversification in the Members of the Nucleophosmin/Nucleoplasmin Family of Nuclear Chaperones

“…Proteins of the NPM3 type include a group of molecular chaperones that are ubiquitously expressed in many tissues, showing the highest levels in pancreas and testis and the lowest in lung, in the case of humans (Shackleford et al 2001). Like NPM1, NPM3 is mainly localized to the nucleolus in interphase cells and active rRNA transcription appears to be required for this localization (Huang et al 2005). NPM3 forms a complex with NPM1 and has been implicated in regulation of NPM1 function in ribosome biogenesis (Huang et al 2005).…”