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* Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, New York, USA; and
Academic Unit of Molecular Vascular Medicine, The Leeds Institute of Genetics Health and Therapeutics University of Leeds, Leeds, UK
1Correspondence: Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., PS 17–401, New York, New York 10032 USA. E-mail: bh2021{at}columbia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of identified variants are targeted to the nonsense-mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the understanding of the gene in the normal and pathological state. —Hudson, B. I., Carter, A. M., Harja, E., Kalea, A. Z., Arriero, M., Yang, H., Grant, P. J., Schmidt, A. M. Identification, classification, and expression of RAGE gene splice variants.
Key Words: RNA splicing • DNA cloning
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, members of the S100/calgranulin family (2)
, and high-mobility group box-1 (HMGB1) (3)
. Binding of these ligands to the receptor results in the activation of a cascade of signal transduction, leading to the expression of proinflammatory and prothrombotic genes (2
, 4)
. The accumulation of these ligands is superimposed with the increased expression of RAGE seen in various pathological states, including cardiovascular disease, diabetic vascular disease, and neuronal degeneration (5
, 6)
. To better understand how RAGE functions in homeostasis and disease, the delineation of the molecular regulation of RAGE at the DNA, mRNA, and protein level will be essential.
The human RAGE gene (also termed AGER) is located on chromosome 6 in the MHC class III region and is composed of a 5' flanking region that regulates its transcription, 11 exons and a short 3'UTR (1
, 7)
. The resulting transcribed mRNA of 1.4 kb is translated into a protein of 404 amino acids with a molecular mass of 55 kDa (1)
. RAGE is composed of a number of distinct protein domains (Fig. 1
); an extracellular region (aa 1–342) composed of a signal peptide (aa 1–22), followed by three immunoglobulin-like domains, including an Ig-like V-type domain, which contains, at least in part, the ligand binding site (aa 23–116) and two Ig-like C2-type 1/2 domains (aa 124–221 and 227–317); a single transmembrane domain (aa 343–363), and a short cytoplasmic tail (aa 364–404). Not unlike other similar members of the immunoglobulin superfamily of receptors, a number of splice variants exist for RAGE (8
9
10
11)
. These result in changes in the amino acid sequence that affect the ligand binding domain and the removal of the transmembrane region, leading to the production of secreted, nonmembrane-bound forms of the receptor (8
9
10
11)
. However, a lack of consistency in these studies remains in terms of what variants were detected, how they were detected, whether these splice variants are biologically relevant, as well as the different nomenclature assigned to the isoforms identified (e.g., secreted forms have been named sRAGE1/2/3, esRAGE, hRAGEsec). Here, we aimed to address these issues by: 1) fully characterizing the 5' and 3' end of the RAGE gene for alternative start/stop sites leading to the production of alternative isoforms; 2) extensively screening tissues and cells for splice variants; 3) classifying and identifying novel forms according to the consensus outlined by the HGNC (12)
; and 4) expressing these variants in vitro to investigate the produced and secreted proteins. We report the identification of numerous novel splice variants and that splicing of the RAGE gene is more extensively complex than previously recognized.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production of aortic smooth muscle cell cytoplasmic cDNA
Human primary aortic smooth muscle cells (AoSMCs) were purchased from Cambrex and maintained in SmGM2 media (Lonza, Basel, Switzerland). Cytoplasmic mRNA was isolated from AoSMC cells by lysing cells with a buffer containing a nonionic detergent to disrupt the plasma membrane and keep the nuclei intact (50 mM Tris, pH 8.0; 140 mM NaCl; 1.5 mM MgCl2; 0.5% Nonidet P-40) (Qiagen). After pelleting nuclei, cytoplasmic RNA was purified using the RNeasy kit (Qiagen). Purified RNA (2.5 µg) was reverse-transcribed using Thermoscript reverse-transcriptase (Invitrogen) and the Generacer Oligo-dT primer.
Screening of splice variants by PCR-restriction endonuclease digestion
Primers were designed to amplify within the 5' and 3' ends of the full-length RAGE gene based on the results of RLM-RACE experiments. The resulting PCR product sequence of the RAGE mRNA to be amplified was analyzed for restriction enzyme recognition sequences using the online tool NEBcutter V2.0 (http://tools.neb.com/NEBcutter2/index.php). Restriction enzyme sites were selected in order to cut the 1246 bp PCR product into a series of bands of differing sizes ranging from 100 to 400 bp. The two restriction enzymes, HindIII and PstI, were chosen from the restriction map to give five bands of easily distinguishable size difference.
A primary PCR was performed on lung and AoSMC cDNA using the RAGE forward (RAGE5'UTR1, 5'-AGGAAGCAGGATGGCAGC-3') and reverse primers (RAGE3'UTR1, 5'-GTCTGAGGCCAGAACAGTTC-3') with Platinum Taq High-Fidelity DNA polymerase system (Invitrogen). The PCR product was diluted 1:500, and a seminested secondary PCR was performed with RAGE5'UTR1 and RAGE reverse-nested primer (RAGE3'UTR2, 5'-TGGGATCTGTCTGTGGGCCCCTCA-3'). The resulting PCR product was purified using the Ultra PCR Clean-Up kit (ABgene) and cloned into the TOPO TA expression vector (pcDNA3.1). Over 100 bacterial colonies were selected as before, and PCR was performed using the RAGE5'UTR1 forward primer and RAGE3'UTR2 reverse primer. The PCR product (5 µl) was electrophoresed on a 1.5% agarose gel to verify product size. The remaining PCR product was digested overnight at 37°C with 10 U of HindIII and PstI and electrophoresed for 2 h on a 3% Midi-Agarose gel (ABgene) at 100 V. PCRs from colonies displaying different restriction patterns from the predicted full-length RAGE were repeated to verify the result. Plasmid DNA was purified from the respective colony as described above, and the resulting DNA sequenced using T7, BGH reverse (pcDNA3.1), and a RAGE-specific primer (RAGE 5'–CTGGGAAGCCAGAAATTGTA-3').
Expression of RAGE splice variants
pcDNA3.1 TOPO clones containing different splice variants were transfected into HEK 293 cells (ATCC, Manassas, VA, USA) to enable expression and characterization of the cloned variant. For transfection, 293 cells were seeded into 6-well dishes at a cell density of 2 x 105 in complete growth medium, and transfections were performed with 2 µg of plasmid DNA using 3 µl of Fugene 6 (Roche, Indianapolis, IN, USA). Conditioned media was collected from the transiently transfected cells after 48 h to analyze for soluble splice variants of RAGE. Cells were lysed using MPER lysis buffer (Pierce, Rockford IL, USA) to characterize cellular variants. Cell lysate (50 µg) or conditioned media (15 µl) was denatured and electrophoresed on 10% Tricine gels (Invitrogen) in Tricine SDS Running Buffer (Invitrogen) for 2 h using the SeeBlue2 and MagicMark protein ladders (Invitrogen) as standards. Proteins were transferred to nitrocellulose membranes (Bio-Rad) in Tris-Glycine Transfer Buffer (Invitrogen) for 1.5 h at 100V. Membranes were blocked in 5% milk-Tris buffered saline Tween-20 (TBST) for 1 h at room temperature and incubated overnight with rabbit anti-RAGE IgG (1)
in blocking solution. Equal loading of protein was assessed by blotting the lysate for GAPDH (Millipore). Detection of proteins was performed using anti-rabbit HRP labeled antibody (Sigma, St. Louis, MO, USA) and visualized by chemiluminescence using the ECL Western blotting Detection System (GE Healthcare, Piscataway, NJ, USA) and Hyperfilm ECL (GE Healthcare).
Bioinformatics
Splice variants of RAGE were identified in Genbank using the BLAST tool (http://www.ncbi.nlm.nih.gov/). Sequence data were viewed and annotated using the BioEdit program (13)
and aligned to RAGE mRNA (M91211) and RAGE DNA (D28769) using Clustalw (http://www.ebi.ac.uk/clustalw/). Protein translation of predicted proteins from splice forms was performed using ExPASy (http://us.expasy.org/tools/dna.html).
Nonsense-mediated mRNA decay (NMD) predictions were made by calculating the distance between the last splice site and the stop codon. The places at which the final splice site was more than 50 bases from the stop codon were designated as putative NMD targets (14)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The majority of clones obtained for the 5' end demonstrated the 5' start site to begin at –11/–13, with –31 and –121 being rarer transcriptional start sites. At the 3' end, only one polyadenylation signal was identified; however, a number of polyadenylation sites were identified a few base pairs apart, at positions +1386, +1388, +1389, and +1390, relative to the ATG (Fig. 2B
). Using this information, primers were designed within the 5'- and 3'-untranslated regions (UTRs) to amplify possible splice variants.
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The identification of RAGE splice variants
To identify different RAGE splice variants, we employed a combination of PCR, cloning, and restriction endonuclease digestion. First, RAGE and its splice variants were amplified from both lung and AoSMC cDNA using primers proximal to the 5' and 3' sites identified above. Second, the resulting mixture of amplification products of RAGE splice forms was cloned into a PCR cloning vector, and 100 individual clones were selected for both lung and AoSMC cDNA. Third, another round of PCR amplification was performed on each clone using the same RAGE primers to amplify the original cDNA. As seen in Fig. 3
A, the canonical human RAGE full-length isoform resulted in a PCR product of 1246 bp. Finally, to distinguish differences in RAGE cDNA due to splicing, restriction endonuclease digestion was performed on the PCR products and band sizes resolved by high-percentage agarose gel electrophoresis. Resolution of differences in undigested PCR product size was obvious for much smaller isoforms (Fig. 3A
); however, the restriction digestion of PCR clones gave five DNA fragments of easily differing size as depicted in the schematic shown in Fig. 3B
. Digestion products comprised RAGE from exon 1–3 (186 bp), exon 3–5 (280 bp), exon 5–6 (110 bp), exon 6–9 (424 bp), and exon 9–3'UTR (246 p). The digestion was designed to enable clear visual differences between RAGE and the previously identified splice variants (8
9
10
11)
as well as to detect potentially novel variants. The digest of the major full-length RAGE isoform and all variants detected in human lung and AoSMC cDNA is shown in Fig. 3C
. Using this approach, we detected a total of 13 alternative splice variants of RAGE from 100 lung and 100 AoSMC selected RAGE-positive clones in addition to the canonical RAGE.
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Characterization of splice variants
Clones that revealed differences by PCR-restriction digestion as outlined above were sequenced in their entirety to identify the potential splice differences. Sequencing of the selected RAGE cDNA clones identified four of the RAGE splice variants that were previously described (including the full-length form of RAGE) (8
9
10
11)
as well as nine additional novel variants as depicted in the schematic shown in Table 1
. Splice variants detected in previous studies included inclusion of part of intron 9 with exon 9 and removal of exon 10, inclusion of intron 1, the entire deletion of exon 8, and deletion of part of exon 3. Sequence data for these splice variants can be found in Genbank, as detailed in Table 1
. Novel isoforms consisted of: inclusion of part of intron 4; inclusion of part of intron 4 and intron 9 with deletion of exon 10; deletion of a part of exon 8; deletion of part of exon 8 and inclusion of intron 9 with deletion of exon 10; deletion of exon 8 and inclusion of intron 9 with deletion of exon 10; and inclusion of intron 9 and deletion of exon 10/11 (see Table 1
). In addition, a number of rare splice variants demonstrating deletion of large numbers of exons were observed, which included the deletion of exons 3–7, exons 2–6, and exons 3–10.
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We then proceeded to classify these and all previously described variants according to the HGNC guidelines, which designate the mRNA splice variants as gene name_vn (where v=mRNA variant and n=number) and the resulting protein as gene name_In (I=protein isoform and n=number), which is shown in Table 1
.
We then assessed the prevalence of RAGE splice variants in the PCR digestions of the 100 lung and 100 AoSMC selected RAGE-positive clones as described above. As shown in Table 2
, in both lung and AoSMC, the canonical human RAGE full-length isoform was the most prevalent variant and accounted for 80% and 70% of detected transcripts, respectively. In lung, the next most prevalent splice form was RAGE_v1, followed by RAGE_v5 and RAGE_v8. All other variants detected in lung were detected only once (RAGE_v4, v6, v10, v11, v12, v13). In AoSMC, RAGE_v1 and RAGE_v2 accounted for 10% of detected transcripts, and RAGE_v5 was the next most prevalent. RAGE_v3, v4, v7, and v9 were detected only once.
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Bioinformatics analysis
Bioinformatic analysis was performed to enable prediction of changes to the protein sequence/function. This resulted in the prediction of both subtle and dramatic changes in the protein sequence of RAGE. RAGE_v4 and RAGE_v5 resulted in a change of amino acid sequence in the extracellular ligand-binding V-domain of RAGE but retention of the reading frame of RAGE, which gave a predicted full-length transmembrane protein. RAGE_v5 introduced 17 new amino acid residues at amino acid number 140 (KVVEESRRSRKRPCEQE), whereas RAGE_v4 led to the loss of 14 amino acids at position 53 (NTGRTEAWKVLSPQ). Inclusion of part of intron 9 (RAGE_v1, RAGE_v4, RAGE_v6, RAGE_v8, RAGE_v9, and RAGE_v10) changed the reading frame of the protein and resulted in the loss of the transmembrane and cytosolic domains of RAGE. Notably, RAGE_v1, which is due to inclusion of part of intron 9 and deletion of exon 10, produced a reading frameshift at amino acid number 332 and created a unique C-terminus sequence (EGFDKVREAEDSPQHM).
Deletion of exon 8, in whole (RAGE_v3) or part (RAGE_v7), also resulted in the loss of the transmembrane and cytosolic domains of RAGE. Using a hydropathy plot, it is predicted that all these variants lack the amino acid motifs required to form a transmembrane protein and, therefore, are likely to produce soluble variants. RAGE_v2, RAGE_v10, RAGE_v11, and RAGE_v12 are predicted to cause major changes in protein sequence. RAGE_v12 produced a short variant, including only part of the V-domain of RAGE; whereas RAGE_v11 produced a short novel protein, with low homology to RAGE (MAAGTAVGACASGGGPIGGGARRWSSSSWWNRNPDL). RAGE_v11 and RAGE_v12 contained a potential downstream initiating ATG codon located at the end of exon 7, which would result in a transmembrane protein lacking the majority of the extracellular domain of RAGE. RAGE_v2 contained a potential downstream, initiating ATG codon located in exon 3, which would result in a transmembrane protein lacking the V-domain of RAGE. RAGE_v13 produced a short protein that lacked most of the extracellular region of RAGE but included the V-domain, the transmembrane region, and cytosolic tail (amino acid numbers 1–94 and 346–404 of RAGE full-length). Bioinformatic analysis using the rule that a stop codon >50 nucleotides upstream of the final exon-exon splice junction inferred that numerous RAGE splice variants are NMD targets. As shown in Table 1
, these candidates include RAGE_v2, RAGE_v3, RAGE_v7, RAGE_v8, RAGE_v9, RAGE_v11, RAGE_v12, RAGE_v14, RAGE_v15, and RAGE_v17.
In vitro expression of splice variants
Splice variants detected in the cytoplasmic extract of AoSMC cells, and thus mature mRNA species, were selected for transfection in human HEK 293 cells directly from the PCR cloning analysis. Transfection of RAGE, RAGE_v1, RAGE_v5, RAGE_v4, RAGE_v7, RAGE_v3, and RAGE_v9 produced proteins of around 55, 48, 58, 45, 50, 40, and 46 kDa, respectively, detectable by Western blotting with polyclonal anti-RAGE IgG, as shown in Fig. 4
. Transfection of RAGE_v2 did not produce a detectable protein by Western blot analysis (Fig. 4A
). Experiments were performed with different RAGE polyclonal or monoclonal commercially available antibodies and with different transfected cell lines (data not shown) to verify the result for RAGE_v2, which still failed to produce a detectable band. Analysis of the conditioned media of the transfected cells confirmed that, as predicted, the RAGE_v1 splice form produced a soluble form, whereas all other forms, including other predicted soluble forms, were detectable only in the cell lysate (Fig. 4A
) and not the conditioned media (Fig. 4B
).
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To further confirm these data, we engineered a truncated RAGE cDNA clone to confirm whether RAGE_v2, previously termed N-truncated RAGE (9)
, forms a mature protein or is a NMD candidate as predicted by the rule that a stop codon >50 nucleotides upstream of the final exon-exon splice junction targets the mRNA species for degradation (14)
. N-truncated RAGE was produced by cloning from exon 3 to the 3'UTR of RAGE as performed by Yonekura et al. (9)
, and, hence, the intron 1 PTC (Fig. 5
A) was removed. As shown in Fig. 5B
, RAGE and N-truncated RAGE constructs resulted in clear bands, whereas RAGE_v2 again failed to produce an immunoreactive band.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. RAGE is also a receptor for at least certain members of the S100/calgranulin family (2)
and HMGB1 (3)
. The diversity of RAGE function may be further enhanced by alternative splicing of its gene, as previously demonstrated (8
9
10
11
, 16)
. However, these studies identified only a small subset of splice forms of RAGE (8
9
10
11
, 16)
. Here, we identified these previously identified isoforms and, in addition, numerous novel variants by extensive screening of human tissue and cells. In the current study, we have classified all the known splice variants of RAGE according to the current nomenclature as proposed by HGNC. We then performed numerous in vitro and bioinformatic analyses to determine the physiologically relevant splice variants.
To fully characterize the splice variants of RAGE, it was essential to identify whether alternative mRNA transcriptional start and stop sites existed. The use of alternative promoters and 3'UTRs is a common feature in the regulation and production of alternative proteins from a single gene locus (17)
. Using RLM-RACE, which selectively isolates and amplifies only 5' capped mature mRNAs, and hence the 5' (start) and 3' (stop) sites of the RAGE gene, we identified a number of very proximal transcription start sites and polyadenylation sites. This finding, therefore, suggests that use of alternative start/stop sites is not a key mechanism in the regulation of RAGE. Hence, we designed our experiments to amplify within the –11 and +1386 positions of RAGE to capture any alternative splice variants of RAGE. To establish consistency between studies, we applied the HGNC nomenclature for splice variants to both previously identified and novel splice forms. Previously identified splicing sites include inclusion of intron 1 (RAGE_v2) (9)
, deletion of part of exon 3 and exon 7 (RAGE_v16) (8)
, inclusion of intron 6 (RAGE_v14) (10)
, and inclusion of intron 9 (RAGE_v1) (9)
. These were all identified in this study, with the exception of the RAGE_v14 splice form. However, it is important to note that the identification of RAGE_v14 was performed using a different tissue source. Thus, these findings may suggest the RAGE_v14 form to be cell- or tissue-specific (9
, 10)
. Analysis of our restriction digestion pattern design suggests RAGE_v14 would have been easy to detect if this was a common variant in the tissue and cell type studied here. This theory is supported by our data in which we have found a different range of splice variants in lung (RAGE_v6, v10, v11, v12, and v13) compared with AoSMC (RAGE_v2, v3, v7, and v9).
Other known variants we detected include the RAGE_v1, previously termed endogenous secretory RAGE (esRAGE) (9)
or sRAGE3 (10)
, which was identified in numerous clones and also in combination with alternative splicing events, including inclusion of intron 4 (RAGE_v6), deletion of exon 8 (RAGE_v9) and deletion of exon 11 (RAGE_v10). RAGE_v1 can be secreted as a "soluble" form from cells in contrast to the other predicted soluble forms (RAGE_v3, v7, and v9); the latter were clearly identified only in cell lysates. Previous studies from Park and colleagues (11)
demonstrated that the RAGE 
ex8 variant was detectable in both cell lysate and conditioned media, in contrast to our data. However, in that study the authors used the pSecTag2 vector, which fuses the inserted protein to the murine Ig 
-chain, which is a leader sequence for protein secretion (11)
. This vector is designed for the expression and secretion of recombinant proteins from transfected cells and may, therefore, artifactually produce secreted soluble proteins that may not normally be secreted in vivo. Further support for our contention that RAGE_v1 is the primary secreted splice variant of RAGE is the analysis of splice variants using the NMD rule. This states that a stop codon >50 nucleotides upstream of the final exon-exon splice junction targets the mRNA species for degradation (14)
and would, therefore, predict that all these splice variants would be targeted to the NMD machinery, except RAGE_v1. Together with our in vitro data, these considerations suggest that RAGE_v1 is the primary splice mechanism resulting in the formation of soluble RAGE.
Recent studies have clearly demonstrated that soluble RAGE can be detected in human blood and that a strong correlation exists between circulating soluble RAGE levels and a range of pathological states, including cardiovascular disease (18
, 19)
, diabetes (19
20
21)
, hypertension (22)
, and dementia/Alzheimer’s disease (23)
. However, these studies used two different ELISA kits to measure soluble RAGE, and these methods have distinct differences. These two assays differ as follows: 1) a generic soluble RAGE ELISA, which measures the total pool of circulating RAGE due the use of polyclonal antibodies against the whole extracellular domain (18)
; and 2) a specific RAGE_v1 ELISA (esRAGE), which detects only this protein and is detected with an antibody raised against the unique C-terminus sequence of RAGE_v1 and does not cross-react with other potential forms of soluble RAGE (24)
. It has been suggested from these studies that, due to the readings with the RAGE_v1/esRAGE ELISA being 4- to 5-fold lower than the generic soluble RAGE ELISA, esRAGE/RAGE_v1 only constitutes a fraction of the circulating pool of soluble RAGE (25
, 26)
. However, no studies have addressed whether these measurements obtained with two different detection methods cross-correlate in the same population of subjects. If RAGE_v1 constitutes only a part of the circulating soluble RAGE pool and RAGE_v1 is the primary splicing cellular mechanism, then the source of the remainder of soluble RAGE and how it is produced remains a key question. It has been suggested that soluble RAGE could be formed by proteolytic cleavage of the membrane bound form from the cell surface (25
, 27)
. However, to date no experimental data support this hypothesis in humans. It is possible that RAGE_v1 constitutes the majority of the circulating pool of soluble RAGE; however, a rigorous ELISA cross-comparison has yet to be performed. Future studies to address these issues are underway.
Apart from these soluble variants of RAGE, other splicing events resulted in extensive predicted changes in the extracellular domain of RAGE. These included changes in the extracellular ligand binding V-domain of RAGE (RAGE_v4, v5) or deletion of the majority of the extracellular domain (RAGE_v2 and v13). These splice variants may have important implications for the regulation and further diversity of ligand binding. RAGE binds multiple ligands in addition to the heterogenous AGEs, such as members of the S100/calgranulin family, HMGB1, and amyloid beta-peptide (15)
. Different isoforms of RAGE with altered ligand binding sites might influence the repertoire of ligands that RAGE can bind, as seen with other similar receptors, including growth factors and cell adhesion molecules (28
, 29)
. Although we could easily detect RAGE_v4 and v5 by Western blot, we were unable to detect the intron 1-containing variant, RAGE_v2 [as demonstrated by Yonekura et al. (9)
to be a40 kDa protein N-truncated RAGE]. NMD analysis predicted the PTC in intron 1 would target this splice variant for degradation and hence could explain why we could not detect any immunoreactive bands for this splice variant. Closer inspection of the cloning strategy used by Yonekura et al. (9)
revealed the authors did not include the full-length intron 1-containing cDNA but a shortened cDNA form starting from exon 3 to the 3'UTR; a sequence common to both RAGE and RAGE_v2. Analysis of the ATG start codon in exon 3 proposed would suggest this also not to be a likely or strong candidate for an initiating codon, according to the Kozak rule (30)
. We, therefore, produced a cDNA truncated form according to the method of Yonekura et al. (9)
. Transfection of the truncated cDNA led to the expression of 40 kDa protein as seen previously. Therefore, two possibilities exist: first, the alternative ATG in exon 3 is switched on when the exon 1 ATG is terminated in intron 1, or second, the RAGE_v2 splice variant is purely a target of the NMD pathway. Our data would, therefore, suggest the second scenario is the more plausible case. The production of N-truncated RAGE is most likely due to the placement of strong transcriptional and translational signals ubiquitous in mammalian expression vectors (30)
. The placement of these stabilizing elements proximal to an ATG codon could most likely lead to the production of a protein and, therefore, together these data suggest RAGE_v2 to be a NMD-targeted splice variant.
We have performed a comprehensive analysis of the RAGE gene and its splice variants in cells and tissue. We have identified numerous novel splice variants and characterized those likely to be functional in health and disease. Further extensive studies are underway to functionally analyze these isoforms, which will help in the understanding of the biology of RAGE.
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ACKNOWLEDGMENTS |
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Received for publication September 24, 2007. Accepted for publication October 25, 2007.
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REFERENCES |
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