Eleanor K. Spicer

The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells.

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.

Plasma Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in MV4-11 Leukemia Cells

AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [32P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [32P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 ± 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of [32P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.

Mechanism of Regulation of bcl-2 mRNA by Nucleolin and A+U-rich Element-binding Factor 1 (AUF1)

The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3′-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3′-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5′ end of the 136-nucleotide bcl-2 AU-rich element (AREbcl-2). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to AREbcl-2. In RNA decay assays, AREbcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of AREbcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

Direct binding of glyceraldehyde 3-phosphate dehydrogenase to telomeric DNA protects telomeres against chemotherapy-induced rapid degradation.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel-shift assays, we show that recombinant GAPDH binds directly with high affinity (K(d)=45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats, and that nucleotides T1, G5, and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA:GAPDH), and GAPDH appears to form a high-molecular-weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active-site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth-inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition; this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.

Regulation of Bcl-2 Expression by HuR in HL60 Leukemia Cells and A431 Carcinoma Cells

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3′-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability. (Mol Cancer Res 2009;7(8):1354–66)

The 3′-untranslated region length and AU-rich RNA location modulate RNA–protein interaction and translational control of β2-adrenergic receptor mRNA

Posttranscriptional controls play a major role in β2-adrenergic receptor (β2-AR) expression. We recently reported that β2-AR mRNA translation is suppressed by elements in its 3′-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with β2-AR mRNA 3′-UTR. In this study, we identified a poly(U) region at the distal end of the 3′-UTR as critical for TIAR binding to β2-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3′-UTR are important determinants that control the translation of β2-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3′-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3′-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3′-UTR length and the ARE RNA location within the 3′-UTR, as key determinants of RNA/protein interactions and translational control of β2-AR mRNA. Further, the importance of 3′-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

Identification of the RNA Binding Domain of T4 RegA Protein by Structure-based Mutagenesis

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H., Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170–1173). Domain I of the RegA protein contains a four-stranded β-sheet and two α-helices. Domain II contains a four-stranded β-sheet and an unusual 3/10 helix. Since β-sheet residues play a role in a number of protein-RNA interactions, one or both of the β-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both β-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of β-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an α-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.

Regulation of nucleolin expression by miR-194, miR-206, and HuR

Nucleolin is a proliferation-associated protein that is overexpressed in multiple types of cancer. The mechanisms leading to overexpression of nucleolin in specific cancers are not fully understood. This study found that nucleolin is notably elevated in breast cancer cell lines MCF-7 and MDA-231 compared to nonmalignant breast epithelial MCF-10A cells. In silico analyses revealed the presence of putative binding sites for microRNAs miR-194 and miR-206 in the 3′-untranslated region (3′-UTR) of Ncl mRNA. Transfection of the three cell lines with pre-miR-194 or pre-miR-206 specifically decreased the Ncl mRNA and protein expression. Treatments of the cells with antagomiR-194 or antagomiR-206 upregulated nucleolin expression ~2- to 3-fold. Co-transfection of cells with a reporter vector containing the Ncl 3′-UTR downstream from the Renilla luciferase gene and pre-miR-194 or pre-miR-206 led to a ~3-fold decrease in Renilla/firefly luciferase activity. Cytoplasmic levels of the RNA-binding protein HuR were higher in MCF-7 and MDA-231 cells than those in MCF-10A cells. RNA immunoprecipitation assays demonstrated that HuR binds to Ncl mRNA in all the three cell types. ShRNA-mediated knock-down of HuR induced a decrease in nucleolin expression, while exogenous expression of HuR led to upregulation of nucleolin expression. Analysis of the polysome–monosome distribution of Ncl mRNA in HuR knock-down cells demonstrated that HuR enhances the translation efficiency of Ncl mRNA. These findings demonstrate that nucleolin expression is down-regulated by miR-194 and miR-206 and upregulated by HuR.

Regulatory mechanism of G protein-coupled receptor trafficking to the plasma membrane: a role for mRNA localization.

Trafficking and localization of G protein-coupled receptors (GPCRs) to the plasma membrane and its retention in the agonist-naive state are critically important for signaling by these receptors. Agonist-induced desensitization of activated GPCRs and their removal from the cell surface have been studied and reviewed extensively. However, less attention has been given to the regulatory mechanisms and different steps that control the trafficking of newly synthesized receptors to the plasma membrane. It is generally believed that the mRNAs encoding GPCRs are targeted to the endoplasmic reticulum by a cotranslational, signal-sequence recognition particle-dependent pathway that results in protein translation and translocation to the plasma membrane. In this chapter, we discuss the importance of cis-targeting elements and trans-recognition factors in GPCR mRNA translational silencing, trafficking, and localization within the cell and its importance in receptor trafficking to the plasma membrane. Knockdown of the critical trans-recognition factors (RNA-binding proteins) resulted in translation of GPCR mRNAs in the perinuclear region and the receptors failed to traffic to the plasma membrane. Thus, a new paradigm is emerging in GPCR trafficking that suggests a fundamental role for mRNA partitioning to specific cytoplasmic regions for efficient plasma membrane localization of the receptors.