QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.014357v1 68/3/816    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ghisolfi, L. Articles by Nicolin, A. Search for Related Content PubMed PubMed Citation Articles by Ghisolfi, L. Articles by Nicolin, A.

Increased Bcl2 Expression by Antisense Oligoribonucleotides Targeting the Adenine-Uridine-Rich Element Motif

Department of Pharmacology, University of Milan, Milan, Italy (L.G., A.B., G.C., G.T., A.N.); and Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy (L.P., A.L., N.S., S.C.)

Received April 29, 2005; accepted June 13, 2005

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
RNA has become a promising target for pharmacological purposes. Most current strategies are directed toward down-regulating its functions. In this study, we provide evidence of the up-regulation of messenger RNA in a sequence-specific manner. The bcl2 (b)-ARE (adenine-uridine-rich element) in the 3'-untranslated region of the b-RNA that regulates the rate of RNA degradation has been targeted with three chemically modified oligoribonucleotides designed in the antisense orientation (asORNs). The three asORNs were studied by a cell-free degradation assay. All three slowed the rate of RNA decay in a dose-response fashion, they were specific to the b-ARE, and two of them were individually effective. asORNs were then transfected into the malignant cells in culture and b-RNA half-life was measured by real-time reverse transcriptase-polymerase chain reaction. We showed that by stabilizing b-RNA the three asORNs increased the expression of b-RNA and of the relevant protein in a dose-response fashion.

A first generation of compounds designed to control gene expression by targeting RNA is represented by the antisense oligodeoxynucleotides (Scherer and Rossi, 2003). The strategy has potential in studies of cell biology (Ravichandran et al., 2004) and for clinical applications (Kalota et al., 2004); antisenses have served to disclose the simplicity of the system and the specificity of action (Schiavone et al., 2004).

Most exciting is RNA silencing, a new field of research that has coalesced during the last decade from independent studies on various organisms (Meister and Tuschl, 2004). The regulatory importance of small noncoding RNA is now recognized, and their mechanism of action is increasingly understood (reviewed in Gottesman, 2004; Storz et al., 2004).

The laboratory exploitation of most of these RNA molecules is focused on decreasing or knocking out gene expression. In contrast with these approaches, we have found that RNAs can be specifically up-regulated, targeting certain motifs such as the degradation elements in cis. In fact, a major control point in gene expression is the turnover of mRNA (Wilusz and Wilusz, 2004).

It is now well established that mRNA decay is not a default process but a tightly regulated process dependent on specific cis-acting sequences and trans-acting factors (Bevilacqua et al., 2003b). An important example of a specific cis-acting element controlling the half-life of mRNA is the adenine-uridine-rich element (ARE) found mainly in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. So far, it represents the most widespread and efficient determinant of RNA (in)stability among those characterized in mammalian cells (Shaw and Kamen, 1986).

In this study, we focused on the bcl2 mRNA (b-RNA) that codes for an antiapoptotic protein involved in neoplastic transformation and/or progression. Bcl2 protein has been shown to lower cell sensitivity to chemotherapy and radiotherapy (Reed et al., 1996; Kroemer, 1997), whereas reduced bcl2 expression has been associated with degenerative disorders, including Parkinson's and Alzheimer's diseases (Saille et al., 1999). In addition to sparing neurons from degeneration (Chen et al., 1997), the rate of bcl2 mRNA degradation might play a role in synapse metabolism.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Schematic structure of b-RNA, core b-ARE nucleotide sequence, and complementary asORNs. Human b-ARE sequence (bases 942-1020, GenBank accession number M14745 [GenBank] ) and three complementary 2'-O-methyl-modified asORNs are depicted.

In previous studies, we described an ARE domain located in the 3'-UTR of b-RNA (b-ARE) that is required for b-RNA decay in vivo (Morelli et al., 1997). We have also shown that the activation of an apoptotic program specifically induces an ARE-dependent b-RNA degradation (Schiavone et al., 2000) and is accompanied by modifications of proteins binding the b-ARE (Donnini et al., 2001).

In the work described here, we used pharmacological tools to inactivate the b-ARE motif within the b-RNA, thereby stabilizing b-RNA and consequently increasing the concentration of the Bcl2 protein in the cells. In particular, we designed 2'-O-methyl oligoribonucleotides (ORNs) in the antisense orientation to b-ARE and used them in an in vitro degradation assay as well as in cultured cells to protect b-RNA from degradation. We showed that antisense ORNs (asORNs) complementary to a critical cis-acting element for b-RNA stability can specifically up-regulate the expression of Bcl2 protein.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Cultures and Chemicals. The cell lines, human follicular t(14,18)-positive B-cell lymphomas DOHH2, promyelocytic leukemia HL60, and neuroblastoma SHSY-5Y, were maintained in RPMI 1640 medium supplemented with 1% glutamine, antibiotics, and 10% fetal calf serum in a humidified atmosphere of 5% CO₂ at 37°C. Viability was monitored by the trypan blue exclusion assay. Three 2'-O-methyl antisense oligoribonucleotides were provided by Dharmacon, Inc. (Lafayette, CO), having been designed based on the ARE core sequence of bcl-2 gene as shown in Fig. 1; a 26-mer degenerated ORN (degORN), 5'-NNNNNNNNNNNNNNNNNNNNNNNNNN-3' (where N is A, G, C, or U), was used as control. The transcription blocker 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) was purchased from Sigma-Aldrich (St. Louis, MO).

Plasmids and In Vitro Transcription. 3'-UTR fragments of bcl-2 mRNA obtained by PCR were cloned in a transcription vector and used for in vitro synthesis of transcripts using SP6 RNA polymerase according to the manufacturer's instructions (Promega, Charbonnières, France), incorporating digoxigenin-labeled NTPs (Roche Diagnostics, Basel, Switzerland) as described previously (Bevilacqua et al., 2003a).

Cell-Free RNA Degradation System and Assays of Inhibition by ORNs. Cell-free RNA degradation assays were performed as described by Bevilacqua et al. (2003a) with minor modifications. Ten micrograms of carrier tRNA and 5 ng of digoxigenin-labeled b-RNA and IGFR-RNA were incubated for 5 min at 80°C after 10 min at 51°C with unlabeled ORNs at the indicated doses before the incubation with cell extracts at 37°C. The relative amount of labeled RNAs was determined by densitometric analysis.

ORN Transfection. AsORNs and degORN at the concentrations described in the text have been delivered with an equal volume of DOTAP Liposomal Transfection Reagent (Roche Diagnostics) to 7.5 x 10⁵ cells/ml according to the manufacturer's instructions.

Measurement of b-RNA Half-Life. At day 1 after ORN transfection, 7.5 x 10⁵ cells/ml were treated with the transcription blocker DRB at a final concentration of 20 µg/ml. At 0, 3, 5, and 7 h after DRB addition, 2 x 10⁶ cells were collected and RNA was extracted for further analysis.

Total RNA Extraction and Real-Time RT-PCR. Total cellular RNA was extracted at the indicated times using the NucleoSpin RNA II columns (Macherey-Nagel, Dueren, Germany) according to the manufacturer's instructions. The RNA was treated with RNase-free DNase (Invitrogen, Carlsbad, CA) and analyzed spectroscopically and by gel electrophoresis for purity and integrity, respectively. For synthesis of cDNA, 0.1 µg/µl total RNA was reverse-transcribed with High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) using standard conditions set by the manufacturer in a total volume of 50 µl. Levels of bcl2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs of each sample were determined by quantitative real-time PCR after TaqMan Universal MasterMix (Applied Biosystems) standard conditions set by the manufacturer; 5 µl of total cDNA was amplified with 2x TaqMan Universal MasterMix Buffer, 20x predeveloped assay reagents system-targeted bcl2, 20x predeveloped assay reagents system control GAPDH, and nuclease-free water (Promega) to a total volume of 25 µl. Each reaction was performed in triplicate for a better statistical reliability of results. The PCR reactions were carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 1 min at 60°C (40 cycles). The amplification plot and cycle threshold data were elaborated with ABI Prism 7000 SDS software (version 1.1). GAPDH data were used to normalize bcl2 cDNA values.

Western Blot Analysis. The assay was performed under standard conditions. In brief, cells were washed twice in ice-cold phosphate-buffered saline and resuspended in 100 µl of ice-cold radioimmune precipitation assay lysis buffer, vortexed for 3 s, and incubated on ice for 30 min. The lysates were centrifuged at high speed for 20 min at 4°C. Equal amounts of proteins were analyzed by 12% SDS-PAGE, blotted onto nitrocellulose membrane Hybond-enhanced chemiluminescence (ECL; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) in a Bio-Rad Trans-blot apparatus at 100 V for 90 min. Blots were processed by an ECL Plus detection kit as instructed by the supplier (Amersham Biosciences). The blots were probed with a mouse anti-Bcl-2 (DakoCytomation Denmark A/S, Glostrup, Denmark) and a rabbit anti-actin (Sigma-Aldrich) antibody followed by a horseradish peroxidase-conjugated secondary antibody.

View larger version (35K): [in this window] [in a new window]   Fig. 2. In vitro b-RNA degradation assays. A, left, three asORNs (total dose, 10 µM) were added to the b-RNA and IGFR-RNA degradation mixture. Lane 1, synthetic transcripts incubated for 60 min without cell extract and without asORNs; lanes 2 to 5, transcripts incubated with cell extracts for the indicated times; lanes 6 to 9, transcripts incubated with cell extract and asORNs for the indicated times; lane 10, transcripts incubated for 60 min without cell extract and with asORNs. Right, time course of decay of b-RNA from the left. B, left, lane 1, synthetic transcripts incubated for 60 min without cell extract and with asORNs; lanes 2 to 5, asORNs; lanes 5 to 9, degORNs. Right, time course of decay of the transcripts from the left; lane 10, transcripts incubated for 60 min without cell extract and with degORNs. C, left, assay conditions as in A. Doses of asORNs added to each tube are indicated. Right, histogram of the dose activity from the left (mean values) obtained by scanning laser densitometry from three assays. Bars indicate the mean ± S.E.

	Results

Top Abstract Materials and Methods Results Discussion References

Inhibition by ORNs of b-ARE Degradation in Cell-Free Assays. The effect of asORNs on the rate of b-ARE degradation was studied in a cell-free system. Transcripts were neither capped at the 5' end with ⁷mGpppG nor polyadenylated at the 3' end, because ARE-directed degradation of an mRNA can occur in a cap-independent fashion (Brewer, 1998; Detich et al., 2001) and in the absence of a poly(A) tract (Chen et al., 2001; Lai and Blackshear, 2001). For assay of decay times, unfractionated cell lysates derived from exponentially growing DOHH2 human lymphoma cells were mixed with the relevant transcripts and incubated at 37°C for various times as described previously (Bevilacqua et al., 2003a).

The rate of b-ARE degradation was significantly reduced by the addition of the asORNs to the degradation mixture, as shown in Fig. 2A. The asORNs exhibited their stabilizing activity selectively on b-RNA and not upon the IGFR control transcript. However, the addition of fully degenerated ORNs (degORNs) did not significantly change the 30-min half-life of b-ARE incubated with cell extracts (Fig. 2B). The half-life of b-ARE was increased to more than 60-min in samples containing the asORNs. Moreover, the inhibition of b-ARE turnover by the asORNs was specific and dose-dependent from 1 to 30 µM (Fig. 2C). The activity of individual asORNs was analyzed at 10 µM under the conditions described above. AsORN2 was the most effective (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   Fig. 3. Inhibition of b-RNA degradation by individual asORNs. Left, assay conditions as in Fig. 2A. asORN 1 was added to reaction mixture in A, asORN2 was added to reaction mixture in B, asORN3 was added to reaction mixture in C at a dose of 10 µM. Right, time course of decay from the left.

SHSY-5Y cells were transfected with a 500 nM concentration of each of the three asORNs. At different times, total RNA was extracted from the cells and amplified by real-time RT-PCR. The mixture of the three asORNs at a total dose of 1.5 µM significantly increased the total amount of b-RNA in the SHSY-5Y cells (Fig. 4A).

View larger version (19K): [in this window] [in a new window]   Fig. 4. b-RNA determination in cells treated with asORNs. A, to a final concentration of 500 nM each, asORNs were delivered with DOTAP liposomal transfection reagent into SHSY-5Y cells. To a final concentration of 1.5 µM, degORNs were given under the same conditions. Cells were untreated or exposed to DOTAP as further controls. RNA obtained from 2 x 106 cells at the indicated times after transfection was analyzed by quantitative real-time RT-PCR. Concentrations of bcl2 were normalized with GAPDH cDNAs. Means ± S.D. from three independent experiments in triplicate are indicated. B, HL60 cells transfected with asORNs as described in A were treated with the transcription inhibitor DRB (20 µg/ml). At the indicated times, b-RNA was analyzed by quantitative RT-PCR and normalized with GAPDH cDNAs. Means ± S.D. from three independent experiments in triplicate are indicated.

Up-Regulation of Bcl2 Protein in Two Cell Lines. The neuroblastoid SHSY-5Y cells were transfected with the three asORNs by DOTAP. The Bcl2 protein was quantified after 5 days of treatment and compared with the amount of Bcl2 in untreated cells and in cells treated with degORNs (Fig. 5A); asORNs induced a 2.5-fold increase in the amount of Bcl2.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Bcl2 protein determinations in cells treated with asORNs. A, asORNs and degORNs were delivered—1.5 µM at day 0 and 0.75 µM at day 3—into SHSY-5Y cells in the same conditions as in Fig. 4A. Left, cell samples were analyzed for Bcl2 protein levels by Western blot assay on days 0, 3, and 5. Right, densitometric histograms of relative band intensities from three independent Western blot experiments. B and C, asORNs and degORNs at the indicated concentrations were given to SHSY-5Y cells (B) or HL60 cells (C) on day 0 and halved on day 3. Left, Western blot of Bcl2 protein on day 5, normalized with -actin in HL60 cells and normalized with -tubulin in SHSY-5Y cells. Right, densitometric histograms of relative band intensities from three independent Western blot experiments.

This work supports the concept that inhibition of b-RNA turnover can increase the expression of the relevant gene at the phenotypic level. It also shows that the inhibition of a subregion within a messenger need not inhibit the activity of other domains.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The concentration of mRNA in cells is a major determinant of the amount of protein expression and phenotype. Although most of the emphasis in the regulation of RNA levels in cells has been placed on transcription, the rate of RNA degradation has recently assumed greater importance (Wilusz and Wilusz, 2004). Fine-tuning of the levels of RNA in the cells through the rapid degradation of redundant products can be achieved by a number of mechanisms (Meyer et al., 2004). cis-Elements can regulate the RNA level at the steady state and, most relevant, "on demand" by particular metabolic conditions or conditions of stress (Kaempfer, 2003).

Identification of the biochemical machinery for RNA degradation has introduced new paradigms and tools of great value for the application in therapeutic pharmacology. RNA displays the unique properties because of its nucleotide sequence, and it can be easily targeted in the cytoplasm.

The work discussed here focuses on the up-regulation of gene expression by inhibition of mRNA decay. The target is the cis-element b-ARE in the 3'-UTR region of the b-RNA. The great sensitivity of b-ARE to physiological or pathological conditions (Lapucci et al., 2002) makes b-ARE an ideal target for regulation of gene expression. Indeed, the last of the findings reported provides experimental evidence that b-RNA degradation can be regulated specifically despite the huge number of different RNA molecules expressed in the cytoplasm.

In this study, we have used exogenous means to inhibit b-ARE activity to slow down the rate of b-RNA degradation and possibly to increase the amount of b-RNA. The final goal, namely the up-regulation of Bcl2 protein, might also be obtained by acting at the level of ARE-binding proteins (AUBPs) that form part of the degradation complex (Raineri et al., 2004). However, these AUBPs recognize a whole set of genes and are thus not specific to a single gene (Chen et al., 2001).

The 2'-O-Me ORNs are believed not to trigger cellular reactions unless properly synthesized to degrade RNA through a small interfering RNA mechanism. It is conceivable that they might hybridize a whole stretch of RNA, causing the temporary inhibition of its functions, possibly through steric hindrance. Therefore, we prepared synthetic ORNs in antisense orientation to b-ARE to decrease the rate of b-RNA degradation.

In a cell-free degradation system, the three designed asORNs significantly slowed the rate of b-RNA degradation. The activity was restricted to b-RNA and was dose-dependent. asORN2 and asORN3 were significantly effective but less potent than when acting together.

The asORN activities were studied in two cell lines transfected with a 500 nM concentration of each asORN. The rate of b-RNA degradation was significantly reduced, as revealed by real-time RT-PCR assay. asORNs doubled the half-life of b-RNA in cells from 3 to 6 h. In a further set of studies, asORNs increased above the basal level, the total amount of b-RNA in the cells. Thus, b-ARE is a suitable target to increase RNA expression in a gene-specific way.

The last set of studies in both HL60 and SHSY-5Y cell lines was carried out to establish whether asORNs are able to up-regulate Bcl2 protein. Although protein amount is finely regulated within cell, asORNs hindering the binding site of degradation promoting AUBPs on b-RNA were able to more than double the amount of Bcl2 protein compared with cells transfected with control degORNs.

All of these findings strengthen the prominent role of AU-rich elements in mRNA decay and depict a new strategy of increasing the expression of a specific gene by acting at the level of a degradation element. The amount of Bcl2 protein was more than doubled relative to the cells transfected with control degORNs. These findings all support the new strategy of increasing the expression of a specific gene by acting at the level of a degradation element. The possibility of up-regulating the concentration of a single protein will surely find therapeutic applications in a variety of diseases or physiological conditions, including those resulting from insufficient production of important factors or abnormal degradation of a particular RNA (Roos et al., 2004).

It is interesting that apoptotic neurons associated with neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, have altered Bcl2 expression (Vyas et al., 1997; Mattson, 2000). Actually Bcl2, in addition to its antiapoptotic functions, has shown differentiative and neurotrophic properties. It can promote dendritic branching and produce regeneration of damaged neurons (Chen et al., 1997). Therefore, Bcl-2 up-regulation represents a strategic goal in neuroprotection therapies (Mattson, 2000).

Such regulation can be achieved by exogenous means as shown here or by transfecting cells with a segment of gene expressing an RNA in antisense orientation as occurring in a human lymphoma and in development (Lee and Roth, 2003; Friedrich et al., 2004). The simplicity of the approach opens new avenues for the regulation of gene expression, because ARE-mRNAs encode a wide range of functionally diverse proteins that belong to different biological processes and are important in several disease states (Khabar, 2005). Although the use of synthetic ORNs may seem to have limited potential in clinical application because ORNs do not cross cell membranes, many efforts are actually underway to overcome this limitation (Soutschek et al., 2004).

	Acknowledgements

 
We thank Dr. P. Woodford for revision of the manuscript.

	Footnotes

 
This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (Milan, Italy), Ministero dell'Istruzione dell'Università e della Ricerca, Consiglio Nazionale delle Ricerche, and Istituto Superiore Sanità (Rome, Italy).

L.G. and L.P. contributed equally to this work.

ABBREVIATIONS: ARE, adenine-uridine-rich element, b, bcl2; b-RNA, 3'-untranslated region of bcl2 mRNA; UTR, untranslated region; ORNs, 2'-O-methyl oligoribonucleotides; asORNs, 2'-O-methyl oligoribonucleotides in antisense orientation; degORNs, 2'-O-methyl degenerated oligoribonucleotides; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; AUBP, ARE-binding protein; RT, reverse transcriptase; IGFR, insulin-like growth factor receptor; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate.

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

	References

Top Abstract Materials and Methods Results Discussion References

 
Bevilacqua A, Ceriani MC, Canti G, Asnaghi L, Gherzi R, Brewer G, Papucci L, Schiavone N, Capaccioli S, and Nicolin A (2003a) Bcl-2 protein is required for the adenine/uridine-rich element (ARE)-dependent degradation of its own messenger. J Biol Chem 278: 23451-23459.[Abstract/Free Full Text]

Bevilacqua A, Ceriani MC, Capaccioli S, and Nicolin A (2003b) Post-transcriptional regulation of gene expression by degradation of messenger RNAs. J Cell Physiol 195: 356-372.[CrossRef][Medline]

Brewer G (1998) Characterization of c-myc 3' to 5' mRNA decay activities in an in vitro system. J Biol Chem 273: 34770-34774.[Abstract/Free Full Text]

Capaccioli S, Quattrone A, Schiavone N, Calastretti A, Copreni E, Bevilacqua A, Canti G, Gong L, Morelli S, and Nicolin A (1996) A bcl-2/IgH antisense transcript deregulates bcl-2 gene expression in human follicular lymphoma t(14;18) cell lines. Oncogene 13: 105-115.[Medline]

Chen CY, Gherzi R, Ong SE, Chan EL, Raijmakers R, Pruijn GJ, Stoecklin G, Moroni C, Mann M, and Karin M (2001) AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107: 451-464.[CrossRef][Medline]

Chen DF, Schneider GE, Martinou JC, and Tonegawa S (1997) Bcl-2 promotes regeneration of severed axons in mammalian CNS. Nature (Lond) 385: 434-439.[CrossRef][Medline]

Detich N, Ramchandani S, and Szyf M (2001) A conserved 3'-untranslated element mediates growth regulation of DNA methyltransferase 1 and inhibits its transforming activity. J Biol Chem 276: 24881-24890.[Abstract/Free Full Text]

Donnini M, Lapucci A, Papucci L, Witort E, Tempestini A, Brewer G, Bevilacqua A, Nicolin A, Capaccioli S, and Schiavone N (2001) Apoptosis is associated with modifications of bcl-2 mRNA AU-binding proteins. Biochem Biophys Res Commun 287: 1063-1069.[CrossRef][Medline]

Friedrich I, Shir A, Klein S, and Levitzki A (2004) RNA molecules as anti-cancer agents. Semin Cancer Biol 14: 223-230.[CrossRef][Medline]

Gottesman S (2004) Small RNAs shed some light. Cell 118: 1-2.[CrossRef][Medline]

Joyce GF (2002) The antiquity of RNA-based evolution. Nature (Lond) 418: 214-221.[CrossRef][Medline]

Kaempfer R (2003) RNA sensors: novel regulators of gene expression. EMBO (Eur Mol Biol Organ) Rep 4: 1043-1047.

Kalota A, Shetzline SE, and Gewirtz AM (2004) Progress in the development of nucleic acid therapeutics for cancer. Cancer Biol Ther 3: 4-12.[Medline]

Khabar KS (2005) The AU-rich transcriptome: more than interferons and cytokines and its role in disease. J Interferon Cytokine Res 25: 1-10.[CrossRef][Medline]

Kroemer G (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3: 614-620.[CrossRef][Medline]

Lai WS and Blackshear PJ (2001) Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail. J Biol Chem 276: 23144-23154.[Abstract/Free Full Text]

Lapucci A, Donnini M, Papucci L, Witort E, Tempestini A, Bevilacqua A, Nicolin A, Brewer G, Schiavone N, and Capaccioli S (2002) AUF1 Is a bcl-2 A + U-rich element-binding protein involved in bcl-2 mRNA destabilization during apoptosis. J Biol Chem 277: 16139-16146.[Abstract/Free Full Text]

Lee LK and Roth CM (2003) Antisense technology in molecular and cellular bioengineering. Curr Opin Biotechnol 14: 505-511.[CrossRef][Medline]

Mattson PM (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1: 120-129.[CrossRef][Medline]

Meister G and Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature (Lond) 431: 343-349.[CrossRef][Medline]

Meyer S, Temme C, and Wahle E (2004) Messenger RNA turnover in eukaryotes: pathways and enzymes. Crit Rev Biochem Mol Biol 39: 197-216.[CrossRef][Medline]

Morelli S, Delia D, Capaccioli S, Quattrone A, Schiavone N, Bevilacqua A, Tomasini S, and Nicolin A (1997) The antisense bcl-2-IgH transcript is an optimal target for synthetic oligonucleotides. Proc Natl Acad Sci USA 94: 8150-8155.[Abstract/Free Full Text]

Raineri I, Wegmueller D, Gross B, Certa U, and Moroni C (2004) Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference. Nucleic Acids Res 32: 1279-1288.[Abstract/Free Full Text]

Ravichandran LV, Dean NM, and Marcusson EG (2004) Use of antisense oligonucleotides in functional genomics and target validation. Oligonucleotides 14: 49-64.[CrossRef][Medline]

Reed JC, Miyashita T, Takayama S, Wang HG, Sato T, Krajewski S, Aime-Sempe C, Bodrug S, Kitada S, and Hanada M (1996) BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem 60: 23-32.[CrossRef][Medline]

Roos C, Sjolinder M, Stenke L, and Tornhamre S (2004) Abnormal LTC4 synthase RNA degradation in neutrophils from CML patients. Br J Haematol 124: 739-745.[CrossRef][Medline]

Saille C, Marin P, Martinou JC, Nicole A, London J, and Ceballos-Picot I (1999) Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid beta-peptide neurotoxicity. Neuroscience 92: 1455-1463.[CrossRef][Medline]

Scherer LJ and Rossi JJ (2003) Approaches for the sequence-specific knockdown of mRNA. Nat Biotechnol 21: 1457-1465.[CrossRef][Medline]

Schiavone N, Donnini M, Nicolin A, and Capaccioli S (2004) Antisense oligonucleotide drug design. Curr Pharm Des 10: 769-784.[CrossRef][Medline]

Schiavone N, Rosini P, Quattrone A, Donnini M, Lapucci A, Citti L, Bevilacqua A, Nicolin A, Capaccioli S (2000) A conserved AU-rich element in the 3' untranslated region of bcl-2 mRNA is endowed with a destabilizing function that is involved in bcl-2 down-regulation during apoptosis. FASEB J 14: 174-184.[Abstract/Free Full Text]

Shaw G and Kamen R (1986) A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659-667.[CrossRef][Medline]

Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature (Lond) 432: 173-178.[CrossRef][Medline]

Storz G, Opdyke JA, and Zhang A (2004) Controlling mRNA stability and translation with small, noncoding RNAs. Curr Opin Microbiol 7: 140-144.[CrossRef][Medline]

Vyas S, Javoy-Agid F, Herrero MT, Strada O, Boissiere F, Hibner U, and Agid Y (1997) Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders. J Neurochem 69: 223-231.[Medline]

Wilusz CJ and Wilusz J (2004) Bringing the role of mRNA decay in the control of gene expression into focus. Trends Genet 20: 491-497.[CrossRef][Medline]

This article has been cited by other articles:

				L. Papucci, E. Witort, A. M. Bevilacqua, M. Donnini, M. Lulli, E. Borchi, K. S. A. Khabar, A. Tempestini, A. Lapucci, N. Schiavone, et al. Impact of Targeting the Adenine- and Uracil-Rich Element of bcl-2 mRNA with Oligoribonucleotides on Apoptosis, Cell Cycle, and Neuronal Differentiation in SHSY-5Y Cells Mol. Pharmacol., February 1, 2008; 73(2): 498 - 508. [Abstract] [Full Text] [PDF]

				A. Bevilacqua, L. Ghisolfi, S. Franzi, G. Maresca, R. Gherzi, S. Capaccioli, A. Nicolin, and G. Canti Stabilization of Cellular mRNAs and Up-Regulation of Proteins by Oligoribonucleotides Homologous to the Bcl2 Adenine-Uridine Rich Element Motif Mol. Pharmacol., February 1, 2007; 71(2): 531 - 538. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.014357v1 68/3/816    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ghisolfi, L. Articles by Nicolin, A. Search for Related Content PubMed PubMed Citation Articles by Ghisolfi, L. Articles by Nicolin, A.