A Circularized Sodium-Calcium Exchanger Exon 2 Transcript
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.8153
ISSN1083-351X
Autores Tópico(s)RNA regulation and disease
ResumoPrevious reports of Na/Ca exchanger gene 1 (NCX1) expression have revealed a major RNA transcript of 7 kilobase pairs (kb), minor transcripts of ∼13 and ∼4 kb, and a relatively abundant 1.8-kb RNA band. In the present report we demonstrate that the 1.8-kb message, which has a tissue and subcellular distribution matching that of full-length NCX1 but is not polyadenylated, corresponds to a perfectly circularized exon 2 species. The circular transcript contained the normal NCX1 start codon, a new stop codon introduced as a consequence of circularization, and encoded a protein corresponding to the NH2-terminal portion of NCX1, terminating just after amino acid 600 in the cytoplasmic loop. A linear version of the circular transcript was prepared and transfected into HEK-293 cells. A protein, matching the predicted size of ∼70 kDa, was expressed, and the transfected cells possessed Na/Ca exchange activity. Although in native tissue we could not detect a protein corresponding exactly to that predicted from the circular transcript, a prominent band of slightly shorter size, possibly representing further proteolytic processing of circular transcript protein, was observed in membranes from LLC-MK2 cells and rat kidney. Previous reports of Na/Ca exchanger gene 1 (NCX1) expression have revealed a major RNA transcript of 7 kilobase pairs (kb), minor transcripts of ∼13 and ∼4 kb, and a relatively abundant 1.8-kb RNA band. In the present report we demonstrate that the 1.8-kb message, which has a tissue and subcellular distribution matching that of full-length NCX1 but is not polyadenylated, corresponds to a perfectly circularized exon 2 species. The circular transcript contained the normal NCX1 start codon, a new stop codon introduced as a consequence of circularization, and encoded a protein corresponding to the NH2-terminal portion of NCX1, terminating just after amino acid 600 in the cytoplasmic loop. A linear version of the circular transcript was prepared and transfected into HEK-293 cells. A protein, matching the predicted size of ∼70 kDa, was expressed, and the transfected cells possessed Na/Ca exchange activity. Although in native tissue we could not detect a protein corresponding exactly to that predicted from the circular transcript, a prominent band of slightly shorter size, possibly representing further proteolytic processing of circular transcript protein, was observed in membranes from LLC-MK2 cells and rat kidney. sodium-calcium exchanger sodium-calcium exchanger gene 1 (SLC8A1) base pair(s) kilobase pair(s) polymerase chain reaction inverse polymerase chain reaction gene-specific primer 1 reverse transcription-coupled PCR 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid guanidine isothiocyanate The sodium-calcium exchanger (NCX)1 plays an important role in the regulation of intracellular Ca2+ levels in a broad number of tissues (1Lederer W.J. He S. Luo S. duBell W. Kofuji P. Kieval R. Neubauer C.F. Ruknudin A. Cheng H. Cannell M.B. Rogers T.B. Schulze D.H. Ann. N. Y. Acad. Sci. 1996; 779: 7-17Crossref PubMed Scopus (25) Google Scholar). Molecular studies of the Na/Ca exchanger have revealed that NCX1 is the predominant Na/Ca exchanger gene and is expressed in almost every tissue but at a particularly high level in heart, brain, and kidney (2Philipson K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Levitsky D.O. Weiss J.N. Ann. N. Y. Acad. Sci. 1996; 779: 20-28Crossref PubMed Scopus (40) Google Scholar). Studies on the organization of the human NCX1 gene have revealed that it comprises at least 14 exons spread out over more than 200 kb of genomic DNA (3Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 4Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar). Several reports have also identified two regions of alternative splicing in NCX1 transcripts from various tissues and animal species. The first site of alternative splicing is in the 5′-untranslated region of the NCX1 message and involves exons referred to as 1a (or 1-Br), 1b, 1c (or 1-Kc), 1d (or 1-Ht), and 1e (3Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 5Nicholas S.B. Yang W. Lee S.L. Zhu H. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar). The use of tissue-specific promoters and splicing patterns involving these exons gives rise to at least three different transcripts, each with a unique exon 1 sequence at the 5′-end (3Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 4Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar, 6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 7Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8Lytton J. Lee S.L. Lee W.S. van Baal J. Bindels R.J. Kilav R. Naveh-Many T. Silver J. Ann. N. Y. Acad. Sci. 1996; 779: 58-72Crossref PubMed Scopus (31) Google Scholar). Studies in rat using an RNase protection assay have demonstrated that heart expresses primarily NCX1 transcripts possessing exon 1-Ht, kidney expresses transcripts with exon 1-Kc, whereas NCX1 transcripts expressed elsewhere contain primarily exon 1-Br (5Nicholas S.B. Yang W. Lee S.L. Zhu H. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar). It is thought that this pattern of splicing may allow independent and selective regulation of NCX1 expression in different tissues. The second region of alternative splicing encodes a stretch of amino acids near the carboxyl terminus of the central intracellular loop of the NCX1 protein. At this site, six different exons (exons 3–8) are arranged in tissue-specific patterns (6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 9Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar, 10Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF PubMed Google Scholar). Heart expresses NCX1 transcripts containing exons 3, 5, 6, 7, and 8, brain expresses transcripts with exons 3, 6, and sometimes 8, and most other tissues express transcripts possessing exons 4 and 6 (and sometimes also 8). The functional consequences of this structural heterogeneity are still uncertain (11Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (199) Google Scholar, 12Ruknudin A. Lederer W.J. He S. Schulze D.H. Biophys. J. 1998; 74 (abstr.): 193Google Scholar). Lying between these two sites of alternative splicing is an unusually long exon 2 sequence (1,832 bp coding for 600 amino acids in human NCX1). Exon 2 encodes the amino-terminal half of the NCX1 protein, including the initiating methionine, the first set of hydrophobic transmembrane segments, and most of the central cytoplasmic regulatory loop. Further alternative splicing, leading to a range of deletions among the carboxyl-terminal transmembrane segments of NCX1, was proposed based on studies in which a 6-kb canine cardiac NCX1 cDNA was expressed in HEK-293 cells (13Gabellini N. Iwata T. Carafoli E. J. Biol. Chem. 1995; 270: 6917-6924Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Nucleotides 3198, 2821, 2620, and 1845 (based on the coordinates of GenBank accession M57523 (14Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (624) Google Scholar)) were identified as potential splice donor sites. None of these sites, however, is close to the exon boundaries identified in the human NCX1 gene (4Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar). Studies of NCX1 expression have all revealed a major transcript of about 7 kb which is expressed abundantly in many tissues (2Philipson K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Levitsky D.O. Weiss J.N. Ann. N. Y. Acad. Sci. 1996; 779: 20-28Crossref PubMed Scopus (40) Google Scholar, 6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar,14Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (624) Google Scholar, 15Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 16Kofuji P. Hadley R.W. Kieval R.S. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 17Yu A.S.L. Hebert S.C. Lee S.-L. Brenner B.M. Lytton J. Am. J. Physiol. 1992; 263: F680-F685PubMed Google Scholar, 18Reilly R.F. Shugrue C.A. Am. J. Physiol. 1992; 262: F1105-F1109PubMed Google Scholar). In addition to this major 7-kb transcript, less abundant transcripts of ∼13 and ∼ 4 kb and an abundant RNA band of 1.8 kb have also been reported (6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 15Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 16Kofuji P. Hadley R.W. Kieval R.S. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 17Yu A.S.L. Hebert S.C. Lee S.-L. Brenner B.M. Lytton J. Am. J. Physiol. 1992; 263: F680-F685PubMed Google Scholar, 18Reilly R.F. Shugrue C.A. Am. J. Physiol. 1992; 262: F1105-F1109PubMed Google Scholar). Although present in many different tissues, the origin of these NCX1 transcripts has not been described. In this report, we describe studies that demonstrate that the 1.8-kb mRNA corresponds to a circularized NCX1 exon 2 transcript encoding a truncated NCX1 protein. The distribution and possible functional role of this transcript are also investigated. All molecular procedures were performed essentially according to standard protocols (19$$Google Scholar, 20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or the directions of reagent manufacturers, unless indicated otherwise. Chemicals were of the highest quality analytical grade available and were obtained from either Fisher, BDH, or Sigma, unless indicated otherwise. Nucleic acid and protein amino acid sequence analysis was performed with the MacVector software package (Oxford Molecular Group) and by connection to the National Center for Biotechnology Information at the National Institutes of Health (www.ncbi.nlm.nih.gov). Total RNA preparations from whole tissues were isolated using the GITC-CsCl centrifugation method and from cultured cells using the GITC acid-phenol extraction protocol. Poly(A)+ mRNA preparations were isolated from total RNA by passage through an oligo(dT) column. To isolate nuclear RNA, nuclei were prepared by hypotonic detergent lysis from LLC-MK2 cells. In brief, cells were collected by centrifugation, washed several times with phosphate-buffered saline, resuspended in 5 × the packed cell volume of hypotonic buffer (10 mm HEPES/KOH, pH 7.9, 0.1 mm EDTA, 0.1 mm EGTA, 1 mmdithiothreitol, and 10 mm KCl), chilled on ice for 15 min, and then lysed by the addition of 0.6% (v/v) Nonidet P-40, followed by gentle mixing and three strokes in a tight-fitting Dounce homogenizer. Nuclei were pelleted from this extract at 1,000 × gfor 3 min, and RNA was isolated using GITC acid-phenol extraction. Samples of RNA were separated on 1% agarose-formaldehyde gels and transferred into a nylon membrane by capillary diffusion overnight. The UV cross-linked membranes were hybridized with antisense digoxigenin UTP-labeled riboprobes according to the directions of the manufacturer (Boehringer Mannheim) as described previously (21Wu K.-D. Lytton J. Am. J. Physiol. 1993; 264: C333-C341Crossref PubMed Google Scholar). Probe A was derived from the 5′-untranslated region exon 1-Kc (nucleotides −375 to −74) of the rat kidney F1 clone as described (6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar). Probe B was prepared from nucleotides −23 to 2871 of the rat kidney F1 clone (spanning exons 2, 4, 6, and 9–12). Probe C was derived from nucleotides 2269 to 2720 of rat kidney NCX1 (spanning most of exon 11 and part of exon 12), prepared as described previously (17Yu A.S.L. Hebert S.C. Lee S.-L. Brenner B.M. Lytton J. Am. J. Physiol. 1992; 263: F680-F685PubMed Google Scholar). The schematic description of the inverse PCR (IPCR) protocol is illustrated in Fig.3 B. 5 μg of RNA from LLC-MK2 cells was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Inc.) using the gene-specific primer GSP1 (GTCTTGGTGGTCTCTCCATT, antisense, nucleotides 346–365; numbering is based on the published canine cardiac NCX1 (14Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (624) Google Scholar), GenBank accession number M36119). The cDNA was converted to second strand essentially as described (21Wu K.-D. Lytton J. Am. J. Physiol. 1993; 264: C333-C341Crossref PubMed Google Scholar) and then purified, phosphorylated, and circularized by ligation in dilute solution. These circles were amplified using the primer pair IPCR-2 (ATGTCCTC[C,T]ATAGAAGTCATCAC, sense, nucleotides 289–311) and IPCR-3 (AGACCATGGCCAC[A,G]AAATACAC, antisense, nucleotides 229–250), which lie upstream from GSP1 and face away from one another. Amplified products were gel purified, subcloned, and sequenced with the Amplitaq FS kit from Perkin-Elmer. Fluorescently labeled sequencing reactions were analyzed at the University of Calgary Core DNA Service Facility. 2 μg of total RNA from LLC-MK2 cells was reverse transcribed using either GSP1 as described above or oligo(dT). The cDNA was then amplified using different pairs of primers based on the exon 2 region. The design of primers is shown in Fig.4 B. The first primer pair, C1 and C1′, face away from one another (C1, same as IPCR-1, above; C1′, GAGTGAGAGCATTGGCATCATG, sense, nucleotides 1638–1659). The second (C2 and C2′) and third (C3 and C3′) primer pairs face toward each other (C2, TTGCTGAGACAGAAATGGAAGGA, sense, nucleotides 92–114; C2′, TCCACAACACCAGGAGAGATGA, antisense, nucleotides 662–683; C3, CTGTCATCTCTCCTGGTGTTGT, sense, nucleotides 659–680; C3′, GAGCTCCAGATGTTCTCAATAC, antisense, nucleotides 1669–1690). The amplified products were gel purified, subcloned, and sequenced. To isolate the circular exon 2 transcript, we designed a 16-nucleotide, 5′-end biotin-labeled, antisense oligonucleotide (circular oligonucleotide: AGAACCTA ACAATTTC) which bridged the circularized 3′- and 5′-ends of exon 2 (8 nucleotides from each end). As a control, we also prepared a similar oligonucleotide (exon 1/2 oligonucleotide, AGAACCTA AGTTTTGA), which spanned the junction of the 3′-end of exon 1 and the 5′-end of exon 2 and was designed to isolate the full-length NCX1 transcript. Total RNA isolated from LLC-MK2 cells (30 μg) was hybridized with 1.5 μmbiotin-labeled circular oligonucleotide or exon 1/2 oligonucleotide and 10 μg of yeast tRNA for 5 min at 65 °C in 300 μl of binding buffer (0.5 m NaCl, 10 mm Tris-Cl, 1 mm EDTA, pH 7.5), cooled slowly to room temperature, and then incubated at 37 °C for 30 min. The samples were then diluted to 2 ml with binding buffer and passed over an Immuno-Pure immobilized monomeric avidin column (Pierce). The procedure followed the instructions from the manufacturer except that phosphate-saline buffer was replaced by binding buffer. The biotin-oligonucleotide-hybridized RNA was eluted from the column in TE buffer (10 mm Tris-Cl, pH 8.0, 1 mm EDTA) containing 2 mm biotin. 10 μg of yeast tRNA was added, and the sample was precipitated with cold ethanol. The precipitated samples were then analyzed by Northern blot with probe B to detect the isolated transcripts. The full-length NCX1 construct was prepared by amplifying oligo(dT)-primed reverse transcribed LLC-MK2 cell total RNA with a pair of primers containing BamHI restriction sites at their 5′-ends (Pr-1, CGCGGATCCAACATGCGGCGATTAAGTCTTTC, sense, nucleotides −3 to 20, and Pr-2, CGCGGATCCCCTTTAGAAGCCTTTTATGTGGC, antisense, nucleotides 2786–2808) using the Expand High Fidelity PCR system from Boehringer Mannheim. A linear version of the circular NCX1 exon 2 transcript was amplified from LLC-MK2 cell total RNA, reverse transcribed with GSP1 (described above), using a pair of primers (Pr-1, described above, and Pr-3, CGCGGATCCGTACACGACACTTCCAACTGT, antisense, nucleotides −24 through −6), which face away from each other. The amplified products were gel purified and cloned into theBamHI site in pcDNA3.1+ (Invitrogen, Inc.). The resulting constructs were confirmed by sequencing. There were three differences between the full-length and truncated proteins, presumably as a consequence of PCR errors: Glu (full-length) for Gly (truncated) at amino acid 39 (counting the initiator Met as 1); Val for Leu-219, and Ser for Phe-482. Transfection of cDNA expression constructs into HEK-293 cells was performed using a standard calcium-phosphate precipitation protocol with BES buffer essentially as described previously (22Tsoi M. Rhee K.-H. Bungard D. Li X.-F. Lee S.-L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1994; 269: 3088-3094Abstract Full Text PDF PubMed Google Scholar). The circular construct cloned in the reverse orientation in the pcDNA3.1 vector was used as a control. Protein expression in crude microsome preparations was analyzed by immunoblotting with the C2C12 monoclonal antibody at 1:1,000 dilution. Calcium transport into transfected HEK cells was analyzed by Fura-2 fluorescent ratio digital imaging essentially as described previously (22Tsoi M. Rhee K.-H. Bungard D. Li X.-F. Lee S.-L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In brief, 2 days after transfection, cells grown on coverslips were loaded by incubation in 5 μm Fura-2/AM, 0.01% pluronic F-127, in serum-free Dulbecco's modified Eagle's medium buffered with 25 mm Tris-HEPES for 20–30 min at room temperature. The coverslips were mounted in a temperature-controlled perfusion chamber (Warner Instruments), maintained at 37 °C, on the stage of a Zeiss Axiovert135 microscope. The cells were perfused continually at 3 ml/min with solutions containing 10 mmTris-HEPES, pH 7.4, 11 mm glucose, 0.5 mmCaCl2, and 145 mm NaCl or LiCl. The 340 nm/380 nm excitation ratio of Fura-2 fluorescence was measured using the ImageMaster System from Photon Technology International. Crude microsome preparations and immunoblotting were performed essentially as described previously (24Lytton J. MacLennan D.H. J. Biol. Chem. 1988; 263: 15024-15031Abstract Full Text PDF PubMed Google Scholar, 25Lytton J. Westlin M. Burk S.E. Shull G.E. MacLennan D.H. J. Biol. Chem. 1992; 267: 14483-14489Abstract Full Text PDF PubMed Google Scholar). In brief, fresh or frozen tissue from rabbit heart, rat heart, or kidney was homogenized with a Polytron in ice-cold sucrose buffer containing a mixture of protease inhibitors (Boehringer Mannheim). LLC-MK2 cells were first swollen hypotonically on ice and then lysed with a Dounce homogenizer in buffer containing protease inhibitors. The cell homogenates were centrifuged, first at 8,000 × g for 20 min to remove nuclei, mitochondria, and debris and then at 100,000 × g for 60 min to pellet a crude fraction containing plasma membranes, endoplasmic reticulum, and other microsomes. These crude microsomal fractions were separated on SDS-polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes, and analyzed by immunoblotting using SuperSignal Plus ECL reagents from Pierce. The monoclonal antibody C2C12 recognizes an epitope between amino acids 372 and 525 (26Porzig H. Li Z. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar) in the central cytoplasmic loop of the exchanger. The monoclonal antibody 6H2, which was a generous gift from Robert Reilly, University of Colorado, recognizes an epitope within the first extracellular 40 amino acids of the mature exchanger protein. Several different groups have reported a relatively abundant NCX1 transcript of ∼1.8 kb in size (15Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 16Kofuji P. Hadley R.W. Kieval R.S. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 18Reilly R.F. Shugrue C.A. Am. J. Physiol. 1992; 262: F1105-F1109PubMed Google Scholar), although in previous studies we had not observed this species (6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 8Lytton J. Lee S.L. Lee W.S. van Baal J. Bindels R.J. Kilav R. Naveh-Many T. Silver J. Ann. N. Y. Acad. Sci. 1996; 779: 58-72Crossref PubMed Scopus (31) Google Scholar, 17Yu A.S.L. Hebert S.C. Lee S.-L. Brenner B.M. Lytton J. Am. J. Physiol. 1992; 263: F680-F685PubMed Google Scholar). Fig.1 shows that we were able to confirm the existence of the 1.8-kb transcript when Northern blots were analyzed with a probe spanning most of the NCX1 coding region (probe B). Indeed, we found that this transcript was present in every tissue and animal species tested. The abundance of the 1.8-kb band appeared to correlate roughly with the abundance of the major 7-kb full-length NCX1 transcript, although the precise ratio varied among animal species. A particularly high amount of 1.8-kb transcript was observed in RNA from monkey tissues and in LLC-MK2 cells, a cell line derived from monkey kidney. To characterize the nature of the 1.8-kb NCX1 transcript, we performed the experiments shown in Fig. 2. First, a Northern blot of total RNA from rat kidney was analyzed with three different probes: A (from exon 1-Kc), B (spanning most of the coding region, exons 2, 4, 6, and 9–12), and C (spanning most of exon 11 and part of exon 12). As seen in Fig. 2 A, the 1.8-kb NCX1 transcript was detected only with probe B, although 7- and ∼13-kb transcripts were clearly evident with all three probes. Next, total RNA isolated from LLC-MK2 cells was passed over an oligo(dT) column to isolate poly(A)+ mRNA. Northern blot analysis of the total RNA, poly (A)+ mRNA, and flow-through fractions revealed that the 1.8-kb NCX1 transcript was present quantitatively in the column flow-through, suggesting that it was not polyadenylated (see Fig. 2 B). Note that the poly(A)+ mRNA lane contained about six times the relative amount of material compared with the other lanes. A band of ∼4.5 kb in size was also visible in the poly(A)+mRNA, as described previously (6Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar). Finally, cellular localization of the 1.8-kb transcript was examined. Total RNA was isolated either from a preparation of detergent-extracted LLC-MK2 cell nuclei or from an equivalent number of whole cells and analyzed by Northern blot. The majority of both 1.8- and 7-kb NCX1 transcripts were present in the cytoplasm and not in the nucleus (see Fig. 2 C). The coincidence of size and the pattern of hybridization with different probes led us to hypothesize that the 1.8-kb NCX1 transcript originated from the unusually long exon 2 sequence. We examined this issue further with a combination of IPCR and RT-PCR studies. Initially, we used the technique of IPCR to define the 5′-untranslated region of NCX1 transcripts expressed in the LLC-MK2 cell line. Following this procedure, as shown in lane 1 of Fig.3 A, two major product bands were detected: one of ∼500 bp and one of ∼1.8 kb. Subcloning and sequencing revealed that the 500-bp band extended back past the beginning of exon 2, ending in unique sequence that we presume to be the NCX1 exon 1 used in LLC-MK2 cells. The sequence of the 1.8-kb fragment also extended to the 5′-end of exon 2 but then continued directly with sequence from the 3′-end of exon 2 (based on the published exon boundaries (3Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 4Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar)). It is noteworthy that the 5′-end of the human NCX1 cDNA reported by Komuro et al. (15Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar) has a structure virtually identical to the LLC-MK2 1.8-kb band, with sequence from the end of exon 2 appearing in the 5′-untranslated region upstream of position −33. Analysis of individual clones for both the 500-bp and 1.8-kb bands indicated that some clones were missing a TAG triplet at the 5′-end of exon 2. This difference may arise from the presence of two closely spaced splice acceptor sites at this location (3Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 4Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar) and is consistent with our previous observation of splicing heterogeneity at the same location in the rat NCX1 gene (5Nicholas S.B. Yang W. Lee S.L. Zhu H. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar). When reverse transcriptase was omitted from the protocol, no bands were detected, as shown in lane 3 of Fig. 3 A, indicating that the amplified products did not arise from genomic DNA contamination. Moreover, when the ligation step of the IPCR protocol was omitted, we still were able to detect the 1.8-kb band, but not the 500-bp product (Fig. 3 A, lane 2). These results suggested the possibility that the 1.8-kb fragment arose from a circularized exon 2 transcript, as illustrated in the schematic of Fig.3 B. To confirm the circular nature of the exon 2 transcript, we performed RT-PCR on LLC-MK2 cell RNA using either a gene-specific primer from exon 2 (GSP1) or oligo(dT) to prime the reverse transcription reaction. The cDNA products were then amplified with different pairs of primers from within exon 2, as shown in panel B of Fig.4. The first pair of primers (C1 and C1′) face away from one another and are therefore expected to amplify only a circular template. The other primer sets (C2 and C2′, C3 and C3′) face toward one another and will thus amplify both linear and circular templates. However, because both of these primer sets have at least one member downstream from the GSP1 reverse transcription priming site, products would only be expected from a linear template if it were primed with oligo(dT). As shown in Fig. 4 A, products were in fact seen from GSP1-primed cDNA for all three primer sets, whereas oligo(dT)-primed cDNA yielded bands only with the primer pairs designed for a linear template. Sequencing confirmed the identity of bands, as illustrated schematically in Fig. 4 B. The IPCR and RT-PCR experiments thus demonstrated the presence of a circularized NCX1 exon 2 transcript of 1.8 kb in length which was not polyadenylated. The 1.8-kb transcript observed on Northern blots was not polyadenylated and was only detected with a probe containing exon 2 sequence. To demonstrate directly that the 1.8-kb transcript corresponded to a circularized exon 2, we used oligonucleotide affinity chromatography. Biotinylated oligonucleotides spanning the junction between the ends of circular exon 2 (circular oligonucleotide) or the 3′-end of exon 1 and the 5′-end of exon 2 (exon 1/2 oligonucleotide) were hybridized with total RNA from LLC-MK2 cells. The hybridized samples were then passed over an avidin column, washed, eluted, and analyzed by Northern blot, as shown in Fig.5. It is evident from these data that hybridization with the circular oligonucleotide selectively enriched for the 1.8-kb transcript, whereas hybridization with the exon 1/2 oligonucleotide selectively enriched for the full-length 7-kb transcript. The circular NCX1 exon 2 transcript contained the normal NH2-terminal start codon for full-length NCX1 and a new stop codon introduced as a consequence of the circularization and thus encoded a protein of 602 amino acids (Fig.6). Constructs expressing the full-length LLC-MK2 cell NCX1, or a linear version of the circular exon 2 transcript encoding the truncated protein, were prepared by high fidelity PCR. The deduced amino acid sequence from these cDNAs is shown in Fig. 6. The full-length monkey kidney NCX1 molecule contained 934 amino acids and was greater than 99% identical to human cardiac NCX1 except in the region of alternative splicing, where human cardiac NCX1 contained exons 3, 5, 6, 7, and 8 (isoform NCX1.1; alternatively spliced exons previously referred to as A, C, D, E and F), whereas LLC-MK2 cell NCX1 contains only exons 4 and 6 (exons B and D, isoform NCX1.3). The truncated protein extends to amino acid 600 of full-length LLC-MK2 cell NCX1, plus two more amino acids (Arg and Phe). Both truncated and full-length constructs were transfected into HEK-293 cells. As seen in the left panelof Fig.7 A, immunoblots using
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