Role Of Nucleolin Research Articles

Abstract 3974: Nucleolin interacts with the microprocessor complex to affect the biogenesis of miR-15a and miR-16-1

Abstract Purpose: To investigate the role of nucleolin in microRNA regulation. Experimental Procedure: Nucleolin was overexpressed and knocked down followed by miRNA expression analysis by qRT-PCR. Nucleolin cellular localization was altered by treatment with parthenolide and verified by cellular fractionation. A change in primary, precursor, and mature microRNA following altered nucleolin localization was determined by endpoint PCR, northern blot, and qRT-PCR, respectively. Immunoprecipitation was utilized to investigate the interaction of nucleolin with the components of the microprocessor complex, Drosha and DGCR8. To determine the interaction of nucleolin with primary microRNA, RNA immunoprecipitation was performed. Results: Altered expression of nucleolin positively correlated with the expression of mature miR-15a and miR-16-1. Upon induction of cytoplasmic nucleolin, mature and precursor miR-15/16 species significantly decreased, while primary species increased. When nuclear levels of nucleolin increased following treatment with parthenolide, miR-15/16 dramatically and significantly increased. However, after nucleolin was knocked down, treatment with parthenolide failed to increase mature miR-15/16. Additionally, primary species were depleted in the presence of nucleolin, indicating increased processing; yet in the absence of nucleolin primary species persisted, indicating a failure to process primary miRNA. Immunoprecipitation studies revealed that nucleolin interacts with both Drosha and DGCR8. This interaction is independent of RNA as it persisted following RNase treatment. RNA immunoprecipitation revealed that nucleolin bound to primary miR-15/16 to a level equal to DGCR8. Conclusions: Nucleolin expression levels do indeed alter miR-15/16 levels. In addition to absolute levels of nucleolin, cellular localization is also important. We conclude that cytoplasmic nucleolin increased primary miRNA while decreasing precursor and mature miRNA due to spatial segregation from the microprocessor complex involved in primary miRNA processing. Increased nuclear nucleolin greatly increased mature miR-15/16 likely by facilitating nucleolin's interaction with the microprocessor complex. Immunoprecipitation data revealed that nucleolin interacts with Drosha and DGCR8 and this interaction is not due to tethering to RNA as it persisted despite RNase treatment. Lastly, nucleolin, which is an RNA binding protein, can bind to primary miRNA. Nucleolin may facilitate processing of primary miRNA by positioning it for cleavage by Drosha, as is nucleolin's role in the biogenesis of ribosomal RNA. Nucleolin is known to localize to the cytoplasm of numerous cancers including CLL and breast cancer. Our data indicates this may lead to a decrease of the tumor-suppressor miR-15/16 providing a survival advantage for cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3974. doi:10.1158/1538-7445.AM2011-3974

A New Paradigm for Aptamer Therapeutic AS1411 Action: Uptake by Macropinocytosis and Its Stimulation by a Nucleolin-Dependent Mechanism

AS1411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-forming oligodeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completely understood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to novel targeted therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for AS1411, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS1411 (FL-AS1411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblasts (resistant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much more efficient than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower in cancer cells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment of various cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in its own uptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required for initial FL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411 uptake at later times. Our results are inconsistent with the previous mechanistic model but confirm that nucleolin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drug delivery.

Abstract 4450: A new paradigm for AS1411 activity: Uptake by macropinocytosis and induction of macropinocytosis by a nucleolin-dependent mechanism

Abstract AS1411 is an experimental drug that is currently in Phase II clinical trials as a novel therapy for cancer. It is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. We have shown previously that AS1411 can efficiently inhibit proliferation and induce cell death in many types of cancer cells, but has little effect on normal cells. Here, we report on the uptake of fluorescently labeled AS1411 (FL-AS1411) in a variety of cancer cell lines (sensitive to AS1411) and non-malignant cells (resistant to AS1411), which we studied using flow cytometry and confocal microscopy. In both cancer and non-malignant cells, uptake of FL-AS1411 was much higher that that of an inactive control oligonucleotide and was rapid (clearly visible in cytoplasmic foci by 15 min). Uptake of FL-AS1411 (evaluated at 2 h) was independent of the presence of serum, but strongly temperature-dependent, indicating an active process. In cancer cells, FL-AS1411 uptake was significantly suppressed by macropinocytosis inhibitor, amiloride, but not by dynamin inhibitor, dynasore, and confocal microscopy showed that AS1411 co-localized mainly with the macropinocytic tracker, dextran, but not with transferrin (a marker of receptor-mediated endocytosis). In contrast, in non-malignant cells, FL-AS1411 uptake was significantly suppressed by dynasore, but not by amiloride. These results suggest that the major mechanism of internalization of AS1411 occurs via macropinocytosis in cancer cells, but via clathrin-dependent and/or caveolae-dependent pathways in normal cells. Additionally, we found that treatment of cancer cells with an oligonucleotide containing the AS1411 sequence caused hyperstimulation of macropinocytosis, provoking an increment in its own uptake. No stimulation of macropinocytosis was observed in non-malignant cells, which are insensitive to AS1411. By using siRNA approaches, we showed that nucleolin is not required for the initial phase of FL-AS1411 uptake (at 2 h), but it is necessary for the induced macropinocytosis and consequently for the second phase of FL-AS1411 uptake (at 24 h). These data confirm the important role of nucleolin in mediating the activity of AS1411 and provide a new model for the mechanism of action of AS1411 in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4450.

Nucleolin associates with the human cytomegalovirus DNA polymerase accessory subunit UL44 and is necessary for efficient viral replication.

In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication

BackgroundNucleolin is a major component of the nucleolus, but is also found in other cell compartments. This protein is involved in various aspects of ribosome biogenesis from transcription regulation to the assembly of pre-ribosomal particles; however, many reports suggest that it could also play an important role in non nucleolar functions. To explore nucleolin function in cell proliferation and cell cycle regulation we used siRNA to down regulate the expression of nucleolin.ResultsWe found that, in addition to the expected effects on pre-ribosomal RNA accumulation and nucleolar structure, the absence of nucleolin results in a cell growth arrest, accumulation in G2, and an increase of apoptosis. Numerous nuclear alterations, including the presence of micronuclei, multiple nuclei or large nuclei are also observed. In addition, a large number of mitotic cells showed a defect in the control of centrosome duplication, as indicated by the presence of more than 2 centrosomes per cell associated with a multipolar spindle structure in the absence of nucleolin. This phenotype is very similar to that obtained with the inactivation of another nucleolar protein, B23.ConclusionOur findings uncovered a new role for nucleolin in cell division, and highlight the importance of nucleolar proteins for centrosome duplication.

Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis

Nucleolin is one of the most abundant protein in the nucleolus and is a multifunctional protein involved in different steps of ribosome biogenesis. In contrast to animals and yeast, the genome of the model plant Arabidopsis thaliana encodes two nucleolin-like proteins, AtNUC-L1 and AtNUC-L2. However, only the AtNUC-L1 gene is ubiquitously expressed in normal growth conditions. Disruption of this AtNUC-L1 gene leads to severe plant growth and development defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer region decondensation, and affects the accumulation levels of pre-rRNA precursors. Remarkably, in Atnuc-L1 plants the AtNUC-L2 gene is activated, suggesting that AtNUC-L2 might rescue, at least partially, the loss of AtNUC-L1. This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.

Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Role of nucleolin in posttranscriptional control of MMP-9 expression

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3′UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin (∼64 kDa form) to the 3′UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells.

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Repression of RNA Polymerase I Transcription by Nucleolin Is Independent of the RNA Sequence That Is Transcribed

Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.