Drosophila Nucleostemin 2 Proved Essential for Early Eye Development and Cell Survival

Human nucleostemin (NS) is a nucleolar protein involved in cell-cycle progression and ribosomal biogenesis. While four NS orthologs have been reported in Drosophila melanogaster, their roles in development have yet to be determined. Here we describe evidence that Drosophila nucleostemin 2 (ns2) plays a significant role in early eye development and is essential for cell survival in vivo.     

Nucleostemin: Another nucleolar “Twister” of the p53-MDM2 loop

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and downregulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.Key words: ribosomal biogenesis, nucleolar stress, nucleostemin, p53, MDM2, cell cycle, cell growth     

Loss of <Emphasis Type="Italic">Drosophila</Emphasis> nucleostemin 2 (NS2) blocks nucleolar release of the 60S subunit leading to ribosome stress

Four nucleostemin-like proteins (nucleostemin (NS) 1–4) were identified previously in Drosophila melanogaster. NS1 and NS2 are nucleolar proteins, while NS3 and NS4 are cytoplasmic proteins. We showed earlier that NS1 (homologous to human GNL3) enriches within the granular components (GCs) of Drosophila nucleoli and is required for efficient maturation or nucleolar release of the 60S subunit. Here, we show that NS2 is homologous to the human nucleostemin-like protein, Ngp1 (GNL2), and that endogenous NS2 is expressed in both progenitor and terminally differentiated cell types. Exogenous GFP-NS2 enriched within nucleolar GCs versus endogenous fibrillarin that marked the dense fibrillar components (DFCs). Like NS1, depletion of NS2 in midgut cells blocked the release of the 60S subunit as detected by the accumulation of GFP-RpL11 within nucleoli, and this likely led to the general loss of 60S subunits as shown by immunoblot analyses of RpL23a and RpL34. At the ultrastructural level, nucleoli in midgut cells depleted of NS2 displayed enlarged GCs not only on the nucleolar periphery but interspersed within the DFCs. Depletion of NS2 caused ribosome stress: larval midgut cells displayed prominent autophagy marked by the appearance of autolysosomes containing mCherry-ATG8a and the appearance of rough endoplasmic reticulum (rER)-derived isolation membranes. Larval imaginal wing disc cells depleted of NS2 induced apoptosis as marked by anti-caspase 3 labeling; loss of these progenitor cells resulted in defective adult wings. We conclude that nucleolar proteins NS1 and NS2 have similar but non-overlapping roles in the final maturation or nucleolar release of 60S ribosomal subunits.     

Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells

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.     

Drosophila GTPase nucleostemin 2 changes cellular distribution during larval development and the GTP-binding motif is essential to nucleoplasmic localization

Nucleostemin (NS), a nucleolar guanosine triphosphate (GTP)-binding protein, plays significant roles in cell cycle progression and ribosomal biogenesis. Drosophila Nucleostemin 2 (NS2), a member of the Drosophila NS family, regulates early eye development and is essential to cell survival in vivo, but the underlying mechanisms have yet to be clarified. Biochemical analysis using the recombinant NS2 protein indicated that NS2 has GTPase activity. Immunohistochemistry revealed that NS2 changes in subcellular locus from the nucleolus to the nucleoplasm during larval development, and that a mutation in the ATP/GTP-binding site motif A (p-loop) prevents nuclear localization of NS2 and results in cytoplasmic distribution. Furthermore, downregulation of NS2 altered the rRNA proportions between the nucleus and the cytoplasm. These results suggest that NS2 at least requires GTP to import into the nucleoplasm.     

A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin

Nucleostemin (NS) was identified as a stem cell- and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra- and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner.     

Nucleostemin

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and down regulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.     

Critical Role of Nucleostemin in Pre-rRNA Processing

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.Nucleostemin (NS)² is a nucleolar protein preferentially expressed in actively proliferating cells. The structure of NS is characterized by two GTP-binding domains, which are involved in the regulation of its dynamic shuttling between the nucleolus and nucleoplasm (1). NS was originally identified as a nucleolar protein prominently expressed in rat neural stem cells and down-regulated during differentiation of these cells in vitro (2). The same authors also found that NS is widely expressed in neural precursor cells in early mouse embryos as well as in a variety of cancer cells and stem cells, including embryonic stem cells and a hematopoietic stem cell-enriched fraction. NS is generally down-regulated in the early stage of differentiation before exit from the cell cycle. In addition, knockdown of NS significantly inhibits proliferation of cortical stem cells and cancer cells. These initial observations led to suggestions that NS is involved in multipotency in stem cells as well as in the regulation of cancer and stem cell proliferation (2).Recent work, however, has demonstrated that NS is in fact widely expressed in many types of normal proliferating cells at levels similar to those in malignant cells. For instance, NS is expressed in normal kidney cells and renal carcinoma cells at comparable levels as detected in histological sections (3). The expression of NS is significantly up-regulated when normal T lymphocytes are activated by concanavalin A (3) and when bone marrow stem cells are stimulated by fibroblast growth factor 2 (4). Cells in NS-null mouse embryos fail to enter the S phase, resulting in embryonic death at the blastocyst stage (5, 6). In early Xenopus embryos NS is also expressed in the sites of active cell proliferation and local depletion of NS results in a decrease in proliferating neural progenitor cells (6). Based on these observations, it was proposed that expression of NS is more closely linked with cell proliferation than with the malignant state or differentiation status of a cell.Several studies have provided evidence that the p53 signaling pathway is involved in the G₁ arrest of the cell cycle induced by the down-regulation of NS. Physical interaction between NS and p53 was initially reported by Tsai and McKay (2). Later, it was shown that the G₁ arrest requires the presence of p53 (7). In the most recent study Dai et al. (8) showed that knockdown of NS enhances the interaction between the p53-binding protein MDM2 and the ribosomal protein L5 or L11, preventing MDM2 from inducing ubiquitylation-based p53 degradation. However, other studies have also suggested that NS may have a p53-independent role in the regulation of cell proliferation. For instance, the depletion of p53 from NS-null blastocysts did not rescue them from the embryonic lethality (6). In addition, NS partial loss-of-function in mouse fibroblasts did not result in any change in the p53 level (5). Furthermore, knockdown of L5 and L11 only partially rescued the G₁ arrest in NS knockdown cells (8). Finally, the fact that NS is primarily localized in the nucleolus, whereas the p53-mediated mechanism occurs in the nucleoplasm, suggests that NS might have an additional role more directly relevant to nucleolar functions.To identify novel functions of NS, we purified an endogenous NS complex from HeLa cell extract and investigated whether NS interacts with other proteins not described previously. Identification of the components of this complex and the alterations of the expression level of NS in HeLa cells led us to uncover a novel role of NS in the processing of rRNA. Our findings not only provide supporting evidence for the hypothesis that NS has a p53-independent function but also demonstrate that NS is critical for ribosome biogenesis, one of the most fundamental processes common for all cell types.     

Modeling and structural analysis of human Guanine nucleotide-binding protein-like 3,nucleostemin

Human GNL3 (nucleostemin) is a recently discovered nucleolar protein with pivotal functions in maintaining genomic integrity and determining cell fates of various normal and cancerous stem cells. Recent reports suggest that targeting this GTP-binding protein may have therapeutic value in cancer. Although, sequence analyzing revealed that nucleostemin (NS) comprises 5 permuted GTP-binding motifs, a crystal structure for this protein is missing at Protein Data Bank (PDB). Obviously, any attempt for predicting of NS structure can further our knowledge on its functional sites and subsequently designing molecular inhibitors. Herein, we used bioinformatics tools and could model 262 amino acids of NS (132-393 aa). Initial models were built by MODELLER, refined with Scwrl4 program, and validated with ProsA and Jcsc databases as well as PSVS software. Then, the best quality model was chosen for motif and domain analyzing by Pfam, PROSITE and PRINTS. The final model was visualized by vmd program. This predicted model may pave the way for next studies regarding ligand binding states and interaction sites as well as screening of databases for potential inhibitors.     

Reactive oxygen species regulate nucleostemin oligomerization and protein degradation

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.     

Nucleostemin和PUMA在骨肉瘤细胞的表达及意义

目的研究Nucleostemin(NS)和p53上调凋亡调控因子(P53 up-regulated modulator of apoptosis,PUMA)在骨肉瘤中的表达。探讨其表达与骨肉瘤的病理分型,临床外科分期的关系。方法收集30例骨肉瘤患者手术标本,采用免疫组织化学SABC法检测这组标本中NS和PUMA的表达,应用图像分析系统测量其阳性表达的平均光密度值。结果 1.NS蛋白定位于细胞核,NS在骨肉瘤组表达高于骨软骨瘤组(P<0.05),在骨肉瘤组随Ennking分期的递增而增加(P<0.05),NS的表达与骨肉瘤的病理分型无关(P>0.05)。2.PUMA定位于细胞质,在骨肉瘤组表达明显低于骨软骨瘤组(P<0.05),其表达与骨肉瘤Ennking分期和病理分型无关(P>0.05)。结论 NS和PUMA在骨肉瘤发生和发展过程中起着重要的作用。     

Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA(+)) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA(+) and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2(ΔBΔM)-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA(+) cells from telomere damage. This work reveals a novel mechanism in telomere protection.     

A nucleostemin-like GTPase required for normal apical and floral meristem development in Arabidopsis

Mammalian nucleostemin (NS) is preferentially expressed in stem cells and acts to promote cell cycle progression. In plants, stem cell activities have to be terminated during flower development, and this process requires the activation of AGAMOUS (AG) gene expression. Here, a nucleostemin-like 1 gene, NSN1, is shown to be required for flower development in Arabidopsis. The NSN1 mRNA was found in the inflorescence meristem and floral primordia, and its protein was localized to the nucleoli. Both heterozygous and homozygous plants developed defective flowers on inflorescences that were eventually terminated by the formation of carpelloid flowers. Overexpression of NSN1 resulted in loss of apical dominance and formation of defective flowers. Expression of the AG gene was found to be up-regulated in nsn1. The carpelloid flower defect of nsn1 was suppressed by the ag mutation in the nsn1 ag double mutant, whereas double mutants of nsn1 apetala2 (ap2) displayed enhanced defective floral phenotypes. These results suggest that in the delicately balanced regulatory network, NSN1 acts to repress AG and plays an additive role with AP2 in floral organ specification. As a midsize nucleolar GTPase, NSN1 represents a new class of regulatory proteins required for flower development in Arabidopsis.     

Nucleostemin is indispensable for the maintenance and genetic stability of hematopoietic stem cells

Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16^Ink4a or p19^Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.     

骨髓间充质干细胞和部分肿瘤细胞中Nucleostemin基因的表达

以分离的人胚胎和大鼠骨髓间充质干细胞 (MSCs) ,6种肿瘤细胞株 ,裸鼠肿瘤和转移瘤组织为实验材料 ,以大鼠心肌组织和人胎盘组织为对照 ,探讨nucleostemin基因的表达情况 .RT PCR结果显示 ,nucleostemin基因在MSCs、肿瘤细胞和肿瘤组织中均有不同程度的表达 ,而大鼠心肌和人胎盘组织中无表达 .DNA测序结果证明 ,扩增的PCR产物与GenBank提供的DNA序列完全同源 .SCID裸鼠肿瘤动物模型定量PCR结果证实 ,nucleostemin的mRNA在裸鼠肿瘤组织和转移瘤组织中表达较高 .研究结果表明 ,在细胞中nucleostemin基因不同水平的表达可能与MSCs、肿瘤细胞的增殖和肿瘤的发生、发展与转移有关 .