Natural antisense RNA inhibits the expression of BCMA, a tumour necrosis factor receptor homologue.
© Hatzoglou et al; licensee BioMed Central Ltd. 2002
Received: 9 January 2002
Accepted: 18 April 2002
Published: 18 April 2002
BCMA (B-cell maturation) belongs to the tumour necrosis factor receptor gene family, and is specifically expressed in mature B lymphocytes. Antisense BCMA RNA is produced by transcription from the same locus and has typical mRNA features, e.g, polyadenylation, splicing, Kozak consensus sequence and an ORF (p12). To investigate the function of antisense BCMA RNA, we expressed BCMA in cell lines, in the presence of antisense p12 or a mutant lacking the initiation ATG codon (p12-ATG).
Overexpression of both p12 and p12-ATG antisense BCMA resulted in a large decrease in the amount of BCMA protein produced, with no change in BCMA RNA levels, indicating that BCMA expression is regulated by antisense BCMA RNA at the translational level. We have also observed slight adenosine modifications, suggestive of the activity of a double-stranded RNA-specific adenosine deaminase.
These data suggest that antisense BCMA may operate under physiological conditions using similar antisense-mediated control mechanisms, to inhibit the expression of the BCMA gene.
BCMA (B-cell maturation) belongs to the tumour necrosis factor receptor (TNFR) gene family , and is specifically expressed in mature B lymphocytes [2, 3]. BCMA and TACI (transmembrane activator and CAML-interactor), another TNFR-homologue , bind two TNF-homologues; BAFF (also called TALL-1, THANK, BLyS, and zTNF4) [5–9] and APRIL . Futhermore, BAFF can bind to a third receptor BAFF-R which is specific to this ligand.[11, 12]. This defines a complexe ligand-receptor system that may play an important role in B lymphocyte differentiation [9, 13–18] and in the regulation of B-cell function.
Naturally occurring antisense RNAs in eukaryotes may regulate sense gene expression, acting at the transcriptional, post-transcriptional or translational level . We have previously reported the presence of a natural antisense BCMA RNA, which is produced by transcription from the same locus as BCMA and has typical mRNA features, e.g. polyadenylation, splicing, Kozak consensus sequence and an open reading frame encoding a 115 amino acid peptide (p12) . RNase protection assays demonstrated the simultaneous presence of natural sense and antisense BCMA transcripts in most of the human B-cell lines and lymphoid tissues tested. We have not succeded to evidence antisense-BCMA transcripts in mouse B lymphocytes. In this study, we investigated whether antisense BCMA RNA regulated the expression of sense BCMA. We observed an inhibition of BCMA expression, the extent of inhibition increasing with the amount of antisense BCMA RNA. This regulation took place at the translational level. These data suggest that similar antisense RNA-mediated control mechanisms may operate in the control of BCMA expression in differenciating primary B lymphocytes.
Results and Discussion
BAFF-R, BCMA and TACI receptors are present on B lymphocytes. BAFF-R binds only BAFF, while BCMA and TACI bind with slight differences in affinity to the same ligands, BAFF and APRIL [13, 15]. Their patterns of signal transduction differ [21, 25], indicating that they may be responsible for different fates of differentiating B lymphocytes. Several mechanisms of control of expression of either one of these receptors may be used by the cell, resulting in different differentiation pathways for B lymphocytes (memory cells, germinal centre cells and/or plasmocytes). In this paper, we present evidence, that in transient expression experiments, BCMA gene expression is tightly and rapidly regulated by antisense BCMA RNA.
The data obtained from the transient expression experiments suggest that antisense BCMA inhibits the expression of BCMA protein and does not affect the expression level of BCMA mRNA. The use of a vector missing the initiation codon for the putative antisense p12 protein showed that the inhibition of the BCMA protein expression is obtained through the action of the antisense RNA and not of the p12 protein. These data suggest that antisense BCMA may operate under physiological conditions using similar antisense-mediated control mechanisms, to inhibit the expression of the BCMA gene
Materials and Methods
Nuclear and cytoplasmic RNAs were isolated by a method described elsewhere . Total RNA was isolated using the TRIzol reagent (Life Technologies, Grand Island, NY) and reverse transcribed using the "Superscript preamplification system" (Life Technologies, Grand Island, NY).
Northern blot analysis
20 μg of total RNA per lane were electrophoresed in a 1% agarose gel, transferred onto Nylon filter (Amersham) and hybridized, as previously described . Probes (human BCMA and β-actin full-lentgh cDNAs) were labelled, using the random prime method . 32P-labelled nucleotides were purchased from Amersham.
Antibodies and reagents
M2 anti-FLAG monoclonal antibody and protease inhibitors were purchased from Sigma-Aldrich. Goat anti-mouse IgG polyclonal antibodies HRP-conjugated and donkey anti-rabbit IgG polyclonal antibodies HRP-conjugated were purchased from Immunotech, Marseille, France. Rabbit polyclonal anti-PI3-K p85 antibodies were from UPI, Lake Placid, NY. Rabbit polyclonal anti-actin antibodies were from Sigma Aldrich. DMEM, fetal calf serum, additional reagents for cell culture, Optimem and Lipofectamine were purchased from Life Technologies.
BCMA 5'ATG (5'-AAGCTTATGTTGCAGATGGCTGGGCA-3')
BCMA 3'TAA (5'-GGATCCTTACCTAGCAGAAATTGATTTC-3')
p12 5' (5'-AAGCTTATGGTTGCGCCTTCCTCCAT-3')
p12 3' (5'-GGATCCTTATTGTAATGCAAGTGTGACCA-3')
p12-ATG 5' (5'-GGAATTCGTTGCGCCTTCCTCCATAG-3')
p12-ATG 3' (5'-TCTAGATTATTGTAATGCAAGTGTGACCA-3')
All primers used in this study were purchased from Genset, Paris, France.
A full length human BCMA (h184) was amplified by PCR from human cDNA, using the BCMA5'ATG and BCMA3'TAA primers. The PCR fragment was digested with BamHI and HindIII restriction enzymes and ligated into the BamHI and HindIII sites of the vector pcDNA3 (Invitrogen, The Netherlands).
N-terminal FLAG-tagged human BCMA was constructed by PCR amplification using the BFL1 and AH7 pair of primers. The PCR product was digested with PstI and BamHI and ligated between the PstI and BamHI sites of the vector pSG5-FLAG .
A full length p12 was amplified by PCR from human cDNA, using the p12 5' and p12 3' pair of primers. The PCR product was digested with HindIII and BamHI restriction enzymes and ligated between the HindIII and BamHI sites of the vector pcDNA3. A mutant lacking the initiation ATG codon (p12-ATG) was amplified by PCR from human BCMA cDNA using the p12-ATG 5' and p12-ATG 3' pair of oligonucleotide primers. The PCR fragment was digested with EcoRI and XbaI restriction enzymes and was ligated between the EcoRI and XbaI sites of the vector pcDNA3.
All expression vectors were constructed by standard recombinant DNA procedures. The sequence of the plasmids constructed by PCR amplification were subsequently verified by dideoxy sequencing (Génome Express, Montreuil, France).
Cell lines and transfections
Human B-lymphocyte cell lines included the precursor REH6 and JEA , the mature Daudi, Raji and the 167 EBV-transformed B lymphoblastoid cell line (obtained from normal cells in the lab) and the myeloma RPMI 8226 cell line. Human embryonic kidney 293 and 293T cells, were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin and were grown at 37°C, in 5% CO2. Adherent cells were seeded in six-well plates (5 × 105 cells per well) in 2 ml of complete medium, incubated at 37°C in 5% CO2 for 20–24 hours and transfected with lipofectamine according to the manufacturer's instructions, using 1 μg of total plasmid DNA, for 6 hours.
Luciferase reporter system for NF-kappaB
The NF-kappaB activation assays were performed using the corresponding luciferase reporter PathDetect Reporting system purchased from Stratagene (La Jolla, CA).
Luciferase and β-galactosidase assays
Transfected cells were washed twice with PBS and lysed with Reporter lysis buffer (Promega, Madison, WI). The luciferase activity was measured using the Reporter assay system (Promega, Madison, WI). β-galactosidase activity was measured using the Luminescent β-galactosidase reporter system (Clontech, Palo Alto, CA). Luminescence was measured using a microplate (EG&G Berthold, Bad Wildbad, Germany). Measurements of luciferase were normalized to β-galactosidase activity and are expressed as a ratio to values obtained from cells treated with vector alone. The relative luciferase activities given are representative of triplicate assays in three independent experiments.
Adherent cells were washed with cold PBS and detached using cold PBS containing 5 mM EDTA and then lysed in lysis buffer (10 mM Tris, pH 7.6, 5 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 400 μM orthovanadate, 1 mM Pefabloc, 5 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin, 0.1% Triton X-100) by incubation for 1 h at 4°C and the supernatant was then clarified by centrifugation. The lysates were analyzed by PAGE and transfered onto PVDF membrane (HybondP, Amersham). The presence of FLAG-tagged BCMA construct was evidenced using M2 anti-FLAG antibody. Rabbit polyclonal anti-PI3K antibody was used to detect the presence of PI3K and rabbit polyclonal anti-actin antibody was used to detect the presence of actin. HRP-conjugated anti-rabbit and anti-mouse IgG and SuperSignal Chemiluminescent substrate (Pierce) were used to reveal the blots.
RNase protection assay
RNase protection assay has been performed as previously described .
This work was funded, in part, by grants from the "Comité Departmental des Hauts de Seine de la Ligue Nationale contre le Cancer", the "Association de Recherche contre le Cancer" (ARC grant 5399), the "Association Claude Bernard" to A.T. and a grant from the Scientific Service of the Embassy of French Republic at Athens, Greece to A.H.
- Madry C, Laabi Y, Callebaut I, Roussel J, Hatzoglou A, Le Coniat M, Mornon JP, Berger R, Tsapis A: The characterization of murine BCMA gene defines it as a new member of the tumor necrosis factor receptor superfamily. Int Immunol. 1998, 10: 1693-1702. 10.1093/intimm/10.11.1693View ArticlePubMedGoogle Scholar
- Laâbi Y, Gras MP, Carbonnel F, Brouet JC, Berger R, Larsen CJ, Tsapis A: A new gene, BCM, on chromosome 16 is fused to the interleukin 2 gene by a t(4;16)(q26;p13) translocation in a malignant T cell lymphoma. EMBO J. 1992, 11: 3897-3904.PubMed CentralPubMedGoogle Scholar
- Gras MP, Laâbi Y, Linares-Cruz G, Blondel MO, Rigaut JP, Brouet JC, Leca G, Haguenauer-Tsapis R, Tsapis A: BCMAp: an integral membrane protein in the Golgi apparatus of human mature B lymphocytes. Int Immunol. 1995, 7: 1093-1106.View ArticlePubMedGoogle Scholar
- von Bulow GU, Bram RJ: NF-AT activation induced by a CAML-interacting member of the tumor necrosis factor receptor superfamily. Science. 1997, 278: 138-141. 10.1126/science.278.5335.138View ArticlePubMedGoogle Scholar
- Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, Ambrose C, Lawton P, Bixler S, Acha-Orbea H, Valmori D, Romero P, Werner-Favre C, Zubler RH, Browning JL, Tschopp J: BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med. 1999, 189: 1747-1756. 10.1084/jem.189.11.1747PubMed CentralView ArticlePubMedGoogle Scholar
- Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, Soppet D, Charters M, Gentz R, Parmelee D, Li Y, Galperina O, Giri J, Roschke V, Nardelli B, Carrell J, Sosnovtseva S, Greenfield W, Ruben SM, Olsen HS, Fikes J, Hilbert DM: BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science. 1999, 285: 260-263. 10.1016/S0925-8388(98)00943-8View ArticlePubMedGoogle Scholar
- Shu HB, Hu WH, Johnson H: TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J Leukoc Biol. 1999, 65: 680-683.PubMedGoogle Scholar
- Mukhopadhyay A, Ni J, Zhai Y, Yu GL, Aggarwal BB: Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem. 1999, 274: 15978-15981. 10.1074/jbc.274.23.15978View ArticlePubMedGoogle Scholar
- Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, Xu W, Parrish-Novak J, Foster D, Lofton-Day C, Moore M, Littau A, Grossman A, Haugen H, Foley K, Blumberg H, Harrison K, Kindsvogel W, Clegg CH: TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature. 2000, 404: 995-999. 10.1038/35010115View ArticlePubMedGoogle Scholar
- Hahne M, Kataoka T, Schroter M, Hofmann K, Irmler M, Bodmer JL, Schneider P, Bornand T, Holler N, French LE, Sordat B, Rimoldi D, Tschopp J: APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J Exp Med. 1998, 188: 1185-1190. 10.1084/jem.188.6.1185PubMed CentralView ArticlePubMedGoogle Scholar
- Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, Hession Schneider, C P, Sizing ID, Mullen C, Strauch K, Zafari M, Benjamin CD, Tschopp J, Browning JL, Ambrose C: BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science. 2001, 293: 2108-2111. 10.1126/science.1061965View ArticlePubMedGoogle Scholar
- Yan M, Brady JR, Chan B, Lee WP, Hsu B, Harless S, Cancro M, Grewal IS, Dixit VM: Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency. Curr Biol. 2001, 11: 1547-1552. 10.1016/S0960-9822(01)00481-XView ArticlePubMedGoogle Scholar
- Thompson JS, Schneider P, Kalled SL, Wang L, Lefevre EA, Cachero TG, MacKay F, Bixler SA, Zafari M, Liu ZY, Woodcock SA, Qian F, Batten M, Madry C, Richard Y, Benjamin CD, Browning JL, Tsapis A, Tschopp J, Ambrose C: BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population. J Exp Med. 2000, 192: 129-136. 10.1084/jem.192.1.129PubMed CentralView ArticlePubMedGoogle Scholar
- Shu HB, Johnson H: B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1. Proc Natl Acad Sci U S A. 2000, 97: 9156-9161. 10.1073/pnas.160213497PubMed CentralView ArticlePubMedGoogle Scholar
- Marsters SA, Yan M, Pitti RM, Haas PE, Dixit VM, Ashkenazi A: Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI. Curr Biol. 2000, 10: 785-788. 10.1016/S0960-9822(00)00566-2View ArticlePubMedGoogle Scholar
- Yu G, Boone T, Delaney J, Hawkins N, Kelley M, Ramakrishnan M, McCabe S, Qiu W-r, Kornuc M, Xia X-Z, Guo J, Stolina M, Boyle WJ, Sarosi I, Hsu H, Senaldi G, Theil LE: APRIL and TALL-1 and receptors BCMA and TACI: system for regulating humoral immunity. Nature Immunol. 2000, 1: 252-256. 10.1038/79802. 10.1038/79802View ArticleGoogle Scholar
- Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, Tschopp J, Browning JL: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999, 190: 1697-1710. 10.1084/jem.190.11.1697PubMed CentralView ArticlePubMedGoogle Scholar
- Khare SD, Sarosi I, Xia XZ, McCabe S, Miner K, Solovyev I, Hawkins N, Kelley M, Chang D, Van G, Ross L, Delaney J, Wang L, Lacey D, Boyle WJ, Hsu H: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S A. 2000, 97: 3370-3375. 10.1073/pnas.050580697PubMed CentralView ArticlePubMedGoogle Scholar
- Vanhee-Brossollet C, Vaquero C: Do natural antisense transcripts make sense in eukaryotes?. Gene. 1998, 211: 1-9. 10.1016/S0378-1119(98)00093-6View ArticlePubMedGoogle Scholar
- Laâbi Y, Gras MP, Brouet JC, Berger R, Larsen CJ, Tsapis A: The BCMA gene, preferentially expressed during B lymphoid maturation, is bidirectionally transcribed. Nucleic Acids Res. 1994, 22: 1147-1154.PubMed CentralView ArticlePubMedGoogle Scholar
- Hatzoglou A, Roussel J, Bourgeade MF, Rogier E, Madry C, Inoue J, Devergne O, Tsapis A: TNF receptor family member BCMA (B cell maturation) associates with TNF receptor-associated factor (TRAF) 1, TRAF2, and TRAF3 and activates NF-kappaB, elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. J Immunol. 2000, 165: 1322-1330.View ArticlePubMedGoogle Scholar
- Devergne O, Hatzivassiliou E, Izumi KM, Kaye KM, Kleijnen MF, Kieff E, Mosialos G: Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation. Mol Cell Biol. 1996, 16: 7098-7108.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagner RW, Yoo C, Wrabetz L, Kamholz J, Buchhalter J, Hassan NF, Khalili K, Kim SU, Perussia B, McMorris FA: Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines. Mol Cell Biol. 1990, 10: 5586-5590.PubMed CentralView ArticlePubMedGoogle Scholar
- Kumar M, Carmichael GG: Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts. Proc Natl Acad Sci U S A. 1997, 94: 3542-3547. 10.1073/pnas.94.8.3542PubMed CentralView ArticlePubMedGoogle Scholar
- Xia XZ, Treanor J, Senaldi G, Khare SD, Boone T, Kelley M, Theill LE, Colombero A, Solovyev I, Lee F, McCabe S, Elliott R, Miner K, Hawkins N, J Guo, Stolina M, Yu G, Wang J, Delaney J, Meng SY, Boyle WJ, Hsu H: TACI Is a TRAF-interacting Receptor for TALL-1, a Tumor Necrosis Factor Family Member Involved in B Cell Regulation. J Exp Med. 2000, 192: 137-144. 10.1084/jem.192.1.137PubMed CentralView ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual, Cold Spring Harbor, NY edn. Plainview, NY: Cold Spring Harbor University Press;. 1989Google Scholar
- Davis LG, Dibner MD, Battey JF: Basic methods in molecular biology. Elsevier, NewYork. 1986Google Scholar
- Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983, 132: 6-13. 10.1016/0003-2697(83)90418-9View ArticlePubMedGoogle Scholar
- Hatzivassiliou E, Cardot P, Zannis VI, Mitsialis SA: Ultraspiracle, a Drosophila retinoic X receptor alpha homologue, can mobilize the human thyroid hormone receptor to transactivate a human promoter. Biochemistry. 1997, 36: 9221-9231. 10.1021/bi963145kView ArticlePubMedGoogle Scholar
- Devergne O, McFarland EC, Mosialos G, Izumi KM, Ware CF, Kieff E: Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression. J Virol. 1998, 72: 7900-7908.PubMed CentralPubMedGoogle Scholar
- Guglielmi P, Davi F: Expression of a novel type of immunoglobulin C lambda transcripts in human mature B lymphocytes producing kappa light chains. Eur J Immunol. 1991, 21: 501-508.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.