Nucleophosmin Is Essential for Ribosomal Protein L5 Nuclear Export

Abstract

Nucleophosmin (NPM/B23) is a key regulator in the regulation of a number of processes including centrosome duplication, maintenance of genomic integrity, and ribosome biogenesis. While the mechanisms underlying NPM function are largely uncharacterized, NPM loss results in severe dysregulation of developmental and growth-related events. We show that NPM utilizes a conserved CRM1-dependent nuclear export sequence in its amino terminus to enable its shuttling between the nucleolus/nucleus and cytoplasm. In search of NPM trafficking targets, we biochemically purified NPM-bound protein complexes from HeLa cell lysates. Consistent with NPM's proposed role in ribosome biogenesis, we isolated ribosomal protein L5 (rpL5), a known chaperone for the 5S rRNA. Direct interaction of NPM with rpL5 mediated the colocalization of NPM with maturing nuclear 60S ribosomal subunits, as well as newly exported and assembled 80S ribosomes and polysomes. Inhibition of NPM shuttling or loss of NPM blocked the nuclear export of rpL5 and 5S rRNA, resulting in cell cycle arrest and demonstrating that NPM and its nuclear export provide a unique and necessary chaperoning activity to rpL5/5S.As the most prominent of subnuclear structures, the nucleolus has long been recognized as the site of active transcription of rRNA and ribosome assembly (8). Various nucleolar proteins, RNAs, and other factors have been implicated in the complex process of ribosome production and maturation (18). Recently, several groups reported the successful isolation and mapping of the mammalian nucleolar proteome (1, 2, 44). While these studies clearly identified proteins and ribonucleoproteins with purported roles in ribosome biogenesis, a surprising number of proteins within the nucleolar proteome (Ͼ100) have no known function. In previous decades, it was assumed that all nucleolar proteins must somehow contribute to static ribosome biogenesis simply by virtue of their localization. However, more-recent findings have demonstrated that the nucleolus is a dynamic subnuclear organelle which regulates numerous cellular processes, prompting a broadened view of the potential functions of nucleolar proteins (28).Nucleophosmin (NPM/B23) is an abundant phosphoprotein that resides within the granular regions of the nucleolus (46). Proliferating cells express NPM at high levels (9, 13), and NPM has been associated with a variety of cellular events, including ribosomal biogenesis, protein chaperoning, and centrosome duplication (13,23,35,36). Structurally, NPM is present in both monomeric and multimeric states, although NPM multimers appear predominant in the nucleolus and may be crucial for the assembly of maturing ribosomes (33,34,53). Furthermore, NPM, along with other nucleolar proteins, is believed (or has been shown) to actively mobilize into distinct subcellular pools, supporting the notion that NPM trafficking may be essential for its (proper) function (6). Indeed, NPM's transit from the nucleolus/nucleus is an essential event in S phase progression; when NPM export was...

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“…The ribosomal L5 protein is a 60S subunit protein that chaperones the 5S rRNA into the nucleolus and out into the cytosol (41). Interestingly, B23 has been shown to directly interact with L5 and play an essential role in its nuclear export (23). Interaction of these 2 proteins in a more general context was reported recently, when B23 was implicated in directing the nuclear export of both 40S and 60S ribosomal subunit proteins (42).…”

“…L5 was previously identified by us as a target protein for p14 (18). It also binds MDM2 in response to nucleolar stress (25), and another p14 target, B23, which is essential for the nuclear export of L5 (23). We attempted to determine whether L5 would be differentially expressed by cells expressing either p14 wt or the various phosphorylation mutants.…”

“…To that effect, p14-expresssing MCF-7 cells were fused with NIH-3T3 cells (murine origin with no MMTV background). The nuclei of these 2 cell types can be visually distinguished (23), so that the export of p14 from human to murine nuclei in the heterokaryon can be monitored. As can be seen in Fig.…”

Nucleocytoplasmic shuttling of BID is involved in regulating its activities in the DNA-damage response

“…Recently, such a mechanism has been demonstrated by showing that nucleophosmin (NPM), which utilizes a conserved CRM1-dependent NES to enable its nucleocytoplasmic shuttling, provides a necessary chaperoning activity for the nuclear export of ribosomal protein L5 (rpL5). 28 Finally, it is tempting to speculate that ATM-mediated phosphorylation of BID regulates its nuclear export. However, this does not seem to be the case, as DNA damage resulted in the nuclear exit of the nonphosphorylatable BID mutant (BID-S61A/S78A-GFP; data not shown).…”

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

“…This may be reminiscent of the acquisition of ribosome binding activity by Mex67 and Mtr2, a function specific to protein loops unique to the yeast proteins and not found in the metazoan proteins (Yao et al, 2007). On the other hand, nucleophosmin has been reported to be required for export of 5S rRNA, and by extension, the 60S subunit, in human cells (Yu et al, 2006). Nucleophosmin does not have an obvious ortholog in yeast.…”