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Department of Pharmacology, University of Milan, Milan, Italy (A.B., L.G., S.F., G.M., A.N., G.C.); Gene Transfer Laboratory, IST, Genoa, Italy (R.G.); and Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy (S.C.)
Received July 30, 2006; accepted October 31, 2006
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Abstract |
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; Moore, 2005; Riddihough, 2005). After the relatively slow process of transcription, the basal level of mRNA is mainly determined by the rate of degradation (Wagner and Lykke-Andersen, 2002; Cougot et al., 2004). Many messengers are further fine-tuned in response to environmental and physiological conditions (Shim and Karin, 2002).
Most inducible genes, including those coding for growth factors, receptors, or regulatory proteins, have unstable mRNAs because cells may have to modulate the corresponding protein level rapidly (Chen and Shyu, 1995). For these genes, the rate of mRNA turnover is the crucial step in regulating basic functions, including cell cycle, cell viability, and reaction to stress (Winzen et al., 2004; Cheadle et al., 2005).
The differences in phenotypes among cells are due mainly to untranslated sequences involved in regulation rather than in coding sequences. Regulatory factors include RNA binding proteins that heavily influence individual's proteome by regulating RNA stability and translation. These proteins associate with multiple messenger RNAs according to a model of coordinated gene expression of functionally related transcripts (Keene and Lager, 2005).
At the level of individual transcript, we have shown that the turnover of the bcl2 messenger can be regulated by the protein Bcl2 itself. A combinatorial mechanism is emerging of RNA regulation by multitarget proteins responsible for different levels of specificity and functionality.
Two major degradation mechanisms have been shown to affect mammalian mRNAs half-life: deadenylation decapping-mediated decay (Gao et al., 2001; Bail and Kiledjian, 2006), and decay mediated by the ARE motives (Guhaniyogi and Brewer, 2001; Raijmakers et al., 2004). In both pathways, gene-specific stretches, usually located in the 3'-UTR and 5'-UTR, are needed for degradation (Stoecklin et al., 2006).
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). These elements have an important role in mRNA stability, being the target site of many AUBPs involved in the decay processes and being able to recruit the exosome (Bevilacqua et al., 2003b; Barreau et al., 2006).
mRNA turnover has been found to be located in discrete cytoplasmic foci, the processing bodies. Under stress conditions that lead to stalled translation, preinitiation complexes can aggregate in cytoplasmic granules from which selected transcripts are degraded (Brengues et al., 2005; Kedersha et al., 2005).
Understanding the molecular mechanisms that regulate the rate of degradation and learning how each mRNA is maintained at the desired level in the cellular cytoplasm are crucial issues. Future studies might unravel new pathogenic pathways leading to many diseases, including cancer (Audic and Hartley, 2004; Denkert et al., 2004) or neurodegenerative disorders (Si et al., 2004) and disclose new therapeutic strategies to regulate gene expression by exogenous means. Moreover, inhibition of the degradation process increases the cellular amount of RNA and of its relative protein.
The regulation of inducible proteins causes obvious phenotypes. For instance, increasing the cellular level of the antiapoptotic protein Bcl2 can protect cells from apoptotic stimuli, although it might have transforming effects (Meijerink et al., 2005). The degradation of Bcl2 mRNA is an ARE-dependent process (Schiavone et al., 2000) mediated by trans- acting elements (Donnini et al., 2004). The rate of Bcl2 mRNA degradation is also dependent on the amount of the Bcl2 protein in the cells (Bevilacqua et al., 2003a).
In the present work, three ORNs designed on the ARE motif (b-ARE) of Bcl2 mRNA have been studied through UV cross-linking assays to prove their AUBP inactivation according to a decoy-aptamer mechanism.
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Materials and Methods |
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-D-ribofuranosyl-benzimidazole (DRB) was purchased from Sigma-Aldrich (St. Louis, MO).
Plasmids, Synthetic Transcripts, and Recombinant Proteins. The KpnI/HindIII 240-base pair fragment containing the SV40 promoter was excised from the pGL3-promoter vector and cloned into the corresponding restriction sites of the pGL4.71 vector (Promega, Madison, WI) to obtain the pGL4.71P plasmid. The 5'-primer CGTCTAGAGTCAACATGCCTGC and the 3'-primer CGTCTAGAGGTGATCCGGCCAA (flanked at the 5'-end by a CG overhang followed by the XbaI restriction site) were used to amplify the 400-base pair segment containing the 3'-UTR ARE sequence from the human bcl2 cDNA fragment. This fragment was cloned into the XbaI cloning site at the 3'-end of the hRlucP gene coding for Renilla reniformis luciferase of the pGL4.71P plasmid to produce the pGL4.71P b-ARE plasmid. Synthetic Bcl2 RNA (b-RNA) (540 nt) used for in vitro degradation assays was prepared as described previously (Bevilacqua et al., 2003a). For UV cross-linking assays, radiolabeled bcl2 transcript was synthesized as described previously (Chen et al., 2001). Proteins GST-KSRP, GST-HuR, and GST-AUF1(p37) were produced in BL21 and purified using glutathione affinity-Sepharose 4B (GE Healthcare, Little Chalfont, Buckinghamshire, UK) resin, whereas histidine-tagged TTP were produced in Escherichia coli and purified using glutathione affinity-Sepharose 4B or nickel-nitrilotriacetic acid (QIAGEN, Valencia, CA).
UV Cross-Linking. UV cross-linking assays were performed as described previously (Chen et al., 2001) with minor modifications. Each AUBP (KSRP, 400 ng; TTP, 300 ng; and HuR or p37-AUF1 200 ng) and 32P-labeled b-RNA (0.5 ng = 2 x 105 cpm) were incubated at room temperature for 20 min in an RNA-binding buffer (20 µl) containing 10 mM HEPES, pH 7.6, 3 mM MgCl2, 100 mM KCl, 2 mM dithiothreitol, 5% glycerol, 0.5% Nonidet P-40, 1 µg of yeast RNA, and 1 µg of heparin. For competition experiments, unlabeled ORNs (1, 10, and 100 µM) were incubated with lysates for 10 min before the addition of the RNA probe. Unbound RNA was digested with RNAase T1 (200 units per reaction) for 15 min at 37°C. Reaction mixtures were transferred to a 96-well plate and irradiated at 4°C for 10 min with a UV Stratalinker (Stratagene, La Jolla, CA). After digestion with RNase A (200 ng per reaction) for 10 min at 37°C, samples were separated by electrophoresis on reducing 10% SDS-polyacrylamide gels. Gels were dried, and 32P-labeled proteins were made visible by autoradiography.
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). The cell-free reaction mixture was maintained for 45 min at 4°C, before the activation at 37°C. Cellular degradation was studied in ORN-treated cells at day 0 and treated with DRB on day 1. After 0, 3, 5, and 7 h, total RNA was extracted with NucleoSpin RNA II columns (Macherey-Nagel, Easton, PA) and amplified by TaqMan real-time RT-PCR (Applied Biosystems, Foster City, CA). The cellular amounts of Bcl2, c-myc, mitogen-activated protein kinase kinase 6 (MKK6), and p27 mRNAs, normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene, were determined as above. Cell Transfection. HEK 293 cells were cotransfected with pcDNA3 and pGL4.71P b-ARE plasmid expressing the chimeric mRNA Rluc-b-ARE by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h, cells were replated in selective media containing 0.8 mg/ml antibiotic G418 (Sigma-Aldrich). Single clones were selected for their resistance to G418 and luciferase activity.
RNA Immunoprecipitation Assay. RNA immunoprecipitation was performed according to Gherzi et al. (2004) with slight modifications. In brief, HEK 293 cells, stably expressing pGL4.71P b-ARE plasmid, were transfected with ORNs at 1.5 µM. After 48 h, cells were lysed in a buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 100 mM NaF, 1 M Na3VO4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 10 mM vanadyl ribonucleoside complex, and 1 mg/ml aprotinin for 15 min at 4°C. Lysates, spun at 14,000g for 15 min at 4°C, and supernatants were incubated overnight with rabbit 
KSRP (Gherzi et al., 2004) or TTP (purchased from Dr. W. Rigby, Dartmouth Medical School, Hanover, NH) or HuR MoAb (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C under rotation and purified with protein A (rabbit antibodies) or Sepharose G (MoAb). RNA, extracted with RNAzol, was determined by real-time RT-PCR using an Rluc-b-ARE chimeric probe.
Western Blot Analysis. The assays were performed under standard conditions. Blots were probed with Bcl2 (Dako North America, Inc., Carpinteria, CA), p27, and HuR (Santa Cruz) MoAbs or rabbit KSRP, TTP, and -actin (Sigma-Aldrich) and detected by ECL Plus kit (GE Healthcare).
Statistics. The data were expressed as means ± S.D., statistical significance was calculated by using a Student's t test, where *, P 
0.05 and **, P 0.01.
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Results |
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Oligoribonucleotides were added at 1 µM to the RNA-AUBP mixture in the reaction tube of the UV cross-linking assay. Figure 1B shows that each ORN inhibited the binding of AUBPs to the b-RNA in a sequence-specific fashion.
ORN2 was the most effective in displacing KSRP, HuR, and AUF1. In the TTP assay, the three ORNs inhibited the binding event with similar efficacy. Although UV cross-linking is only a semiquantitative assay, ORNs were able to displace AUF1, an effective regulator of the b-RNA half-life (Lapucci et al., 2002), in a dose-response fashion (Fig. 1C). To address the displacement activity in cells, HEK 293 cells stably transfected with pGL4.71P b-ARE plasmid expressing chimeric mRNA Rluc-b-ARE were treated with ORNs (1.5 µM). RNA immunoprecipitation assays were performed 30 h after transfection with ORNs to evaluate the effect on AUBPs binding to b-ARE. The amount of the relevant RNA was significantly reduced in cells treated with sense ORNs, as shown in Fig. 1D.
In Vitro Inhibition of b-RNA Degradation. Whether displacement of AUBPs might change the rate of b-RNA turnover was analyzed by a cell-free degradation assay. The b-RNA fragment including b-ARE, labeled by incorporation of the precursor DlG-UTP, was transcribed as reported previously (Bevilacqua et al., 2003a). An IGFR RNA fragment, labeled as above, was added to the degradation mixture as a control. For the decay assays, cell lysates from DOHH2 lymphoma cells were mixed with the relevant transcripts and incubated at 37°C for the indicated times.
The three ORNs, used in combination to span the entire b-ARE motif at a final concentration of 10 µM, proved highly effective (Fig. 2A). Figure 2B shows that the half-life of the b-RNA was more than twice that of samples containing degORNs. In contrast, the degradation rate of the IGFR transcript, not expressing ARE motifs, was unaffected. The slowdown of b-RNA turnover by the ORNs was dose-dependent: the activity increased from 1 to 10 µM and decreased at higher doses (Fig. 2, C and D). Oligonucleotides designed in between ORN1 and ORN2 or ORN2 and ORN3 or flanking the core sequence did not display a significant activity (data not shown).
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Increased Level and Stabilization of RNAs in Cell Lines. Raji lymphoma cells and HL60 promyelocytic cells were exposed to three ORNs mixed to a total concentration of 1.5 µMin N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate. The amount of Bcl2 mRNA in the cells was measured by real-time RT-PCR (TaqMan Probe System) at the indicated times.
As shown in Fig. 4, A and C, ORNs were able to increase the amount of Bcl2 mRNA in both cell lines, with the highest activity being obtained 48 h after treatment. The possibility that ORNs might stabilize Bcl2 mRNA was studied by analyzing the amount of Bcl2 mRNA in extracts from cells treated with the transcription inhibitor DRB. Using the real-time RT-PCR, the Bcl2 mRNA half-life was calculated as 4.5 h in untreated cells and as more than 7 h in ORN-treated Raji cells (Fig. 4B); it was 3.5 h in untreated versus 6 h in ORN-treated HL60 cells (Fig. 4D). Individual AUBPs can interact and regulate the mRNA decay of many genes (Chen and Shyu, 1995); it is thus possible that the AUBPs displacement can stabilize ARE-expressing genes other than bcl2 (Supplemental Data S1). Therefore, we measured the amount of c-myc RNA in HL60 cells (Fig. 4E) and the amount of MKK6 and p27 RNA in Raji cells (Fig. 4, F and G). The amount of all three RNAs was significantly increased in cells treated with sense-oriented ORNs.
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Discussion |
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; Nimjee et al., 2005). We also examined whether sense-oriented ORNs would change the rate of b-RNA degradation in in vitro assays and in viable cells. In direct UV cross-linking assays, ORNs successfully competed with the b-ARE motif, probably inhibiting the interaction of recombinant HuR, KSRP, AUF1, or TTP with b-RNA. Although both stabilizing and destabilizing AUBPs were displaced by oligonucleotides, RNA stabilization turned out as the final effect of ORN treatment.
ORN2, designed on the central region of the ARE motif, was the most effective to inhibit the binding of all four AUBPs. In this subregion are located relevant determinants for the degradation machinery, determinants that are required for the binding of important AUBPs. The binding activity of HuR and KSRP to the b-ARE was strongly decreased also in HEK 293 cells upon ORNs treatment, as shown by RNA immunoprecipitation.
Each AUBP, including those studied here, can bind AREs present in several mRNAs (Good, 1995; Lu and Schneider, 2004). How one AUBP can bind different nucleotide structures is not yet known. A possibility is the recognition of characterizing elements such as the AUUUA pentamer or U-rich stretches expressed in the 3'-UTR of many messengers (Winzen et al., 2004; Fialcowitz et al., 2005). According to the post-transcriptional operon model (Keene and Lager, 2005), RNA binding proteins coordinately regulate the expression of multiple mRNAs encoding functionally related proteins. These mechanisms provide combinatorial regulation of genetic information, including the evolution of multifunctionality of eukaryotic proteins. Systems and high through put approaches have provided insight into coordinated post-transcription regulation of gene expression. Multiple interactions have been shown by micro-RNAs, which can also complement short 3'-UTR stretches (5-8 nt) within a variety of RNA targets contributing to regulate protein synthesis along a coordinated fashion (Yoon and De Micheli, 2005; Kim and Nam, 2006). This study, although not involved directly into this matter, suggests that characterizing structures expressed in most AREs may behave as shared targets for AUBPs.
The binding of a protein to different AREs of unrelated transcripts was inhibited by sequence-specific ORNs mimicking endogenous structures. Meanwhile, one single ORN can compete with different AUBPs.
The decoy activity in vitro and in cell lines actually turned out to regulate the rate of b-RNA degradation. In the cell-free system, ORNs protected b-RNA from the degradative action of an effective cell extract. The most effective individual ORN was ORN2, although ORN3 showed no detectable activity. Thus, the stabilizing activity mirrored the decoy activity. Moreover, the effect of the triple ORN mixture on the rate of b-RNA degradation was dose-dependent. This suggests that the decreased interaction of destabilizing AUBPs with Bcl2 mRNA transcript, due to the ORN presence, leads to an mRNA stabilization.
In different human cell lines, ORNs were able to significantly increase the level of Bcl2 mRNA. The biochemical mechanism might be dependent on the ORN ability to slow down the rate of degradation, as shown by the time course determinations of the Bcl2 mRNA in cells treated with a transcription inhibitor.
Not entirely unexpected, ORNs also inhibited the rate of degradation of unrelated mRNAs whose decay is dependent on AREs and AUBPs. These observations might figure out the coordinated and temporally linked regulation of functionally related transcripts.
Consistent with the increased amount of mRNA, the level of Bcl2 protein was significantly and dose-dependently higher in cells exposed to ORNs. Moreover, as predicted above, the level of proteins encoded by stabilized RNAs were increased as well. It is shown that the p27 protein, which fluctuates during the cell cycle often in coordination with the level of Bcl2 (Calastretti et al., 2001; Greider et al., 2002), was also up-regulated. Although the mechanism of RNA augmentation by ORNs homologous to the b-ARE motif is only in part understood, the findings described here are in keeping with a model in which ARE is regulated at different levels of specificity and coordination (Keene and Lager, 2005).
The ARE of the bcl2 gene, characterized by a unique nucleotide sequence, might be targeted specifically by the relevant protein that actually regulates the rate of degradation of its own RNA, according to a negative feedback mechanism (Bevilacqua et al., 2003a). The bcl2 transcript can also be regulated specifically by antisense-oriented oligoribonucleotides targeting the b-ARE motif (Ghisolfi et al., 2005), supporting the concept of the individual regulation.
Conversely, the coordinated regulation of multiple related transcripts, as shown here by the use of sense-oriented ORNs, is not in contrast with the single transcript regulation. On the basis of studies on AUBP displacement, sequence-specific ORNs, by competing for the docking sites of proteins that recognize common elements within the ARE structures, might exert a multitranscript activity. These findings support a combinatorial modality for the coordinating transcript regulation based on the interaction of trans-acting factors and cis-elements.
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Acknowledgements |
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Footnotes |
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A.B. and L.G. contributed equally to this work.
ABBREVIATIONS: ARE, adenine-uridine rich element; AUBP, adenine-uridine binding protein; ORN, oligoribonucleotide; UTR, untranslated region; b-ARE, bcl2 adenine-uridine rich element; b-RNA, 3'-untranslated region of Bcl2 RNA; HEK, human embryonic kidney; degORNs, 2'-O-methyl-degenerated oligoribonucleotide; DRB, 5,6-dichloro-1--D-ribofuranosyl-benzimidazole; GST, glutathione transferase; RT-PCR, reverse transcription polymerase chain reaction; MoAb, monoclonal antibody; IGFR, insulin-like growth factor-I receptor; nt, nucleotide MKK6, mitogen-activated protein kinase kinase 6; Rluc, Renilla reniformis luciferase.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Angelo Nicolin, Department of Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. E-mail: angelo.nicolin{at}unimi.it
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, untreated; 
, degORNs; 
, sense ORNs. Equal amounts of HuR, KSRP, or TTP were detected in ORNs of treated and untreated samples, as shown in Western blots (bottom). The data are representative of one of three independent experiments.
