Intergenic mRNA Molecules Resulting fromtrans-Splicing
2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês
10.1074/jbc.m109175200
ISSN1083-351X
AutoresCsaba Finta, Peter G. Zaphiropoulos,
Tópico(s)RNA modifications and cancer
ResumoAccumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, andCYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 orCYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 andCYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules,i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be ∼0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2–13 of CYP3A4 revealed catalytic activity toward testosterone. Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, andCYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 orCYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 andCYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules,i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be ∼0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2–13 of CYP3A4 revealed catalytic activity toward testosterone. The ongoing genome projects are allowing the determination of the coding potential of several eukaryotic species at a rapidly increasing pace. However, as most eukaryotic genes are in pieces, consisting of discrete exonic sequences flanked by noncoding intronic segments, defining the expressed fraction of the genetic material is obviously of highest priority. The puzzling problem of identifying relatively short exons in a vast excess of intronic sequences is further complicated by recent results indicating that, during gene expression, an additional level of complexity also exists. Specifically, in several higher eukaryotes, processed RNA does not always represent a simple linear combination of exonic sequences. Thus, in addition to canonical transcripts, gene expression may also result in scrambled RNA molecules in which exons juxtapose in an order that is different to that in the gene (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar, 2Cocquerelle C. Daubersies P. Majerus M.A. Kerckaert J.P. Bailleul B. EMBO J. 1992; 11: 1095-1098Crossref PubMed Scopus (237) Google Scholar, 3Zaphiropoulos P.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6536-6541Crossref PubMed Scopus (204) Google Scholar, 4Zaphiropoulos P.G. Mol. Cell. Biol. 1997; 17: 2985-2993Crossref PubMed Scopus (148) Google Scholar, 5Marcucci G. Strout M.P. Bloomfield C.D. Caligiuri M.A. Cancer Res. 1998; 58: 790-793PubMed Google Scholar, 6Caldas C., So, C.W. MacGregor A. Ford A.M. McDonald B. Chan L.C. Wiedemann L.M. Gene (Amst.). 1998; 208: 167-176Crossref PubMed Scopus (68) Google Scholar, 7Chao C.W. 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There are four known CYP3Agenes in humans, CYP3A4, CYP3A5,CYP3A7, and the recently discovered CYP3A43(25Domanski T.L. Finta C. Halpert J.R. Zaphiropoulos P.G. Mol. Pharmacol. 2001; 59: 386-392Crossref PubMed Scopus (197) Google Scholar, 26Gellner K. Eiselt R. Hustert E. Arnold H. Koch I. Haberl M. Deglmann C.J. Burk O. Buntefuss D. Escher S. Bishop C. Koebe H.G. Brinkmann U. Klenk H.P. Kleine K. Meyer U.A. Wojnowski L. Pharmacogenetics. 2001; 11: 111-121Crossref PubMed Scopus (214) Google Scholar, 27Westlind A. Malmebo S. Johansson I. Otter C. Andersson T.B. Ingelman-Sundberg M. Oscarson M. Biochem. Biophys. Res. Commun. 2001; 281: 1349-1355Crossref PubMed Scopus (173) Google Scholar). The proteins are very similar to each other bearing 71–88% amino acid identities. All four genes consist of 13 exons and form a cluster on chromosome 7q21–22.1. Interestingly, comparison of GenBank™ entries encompassing various CYP3A genomic sequences revealed a rather unique arrangement of the CYP3Alocus, specifically that the CYP3A43 gene is in a head-to-head orientation with the other three, CYP3A4,CYP3A7, and CYP3A5, genes (Ref. 25Domanski T.L. Finta C. Halpert J.R. Zaphiropoulos P.G. Mol. Pharmacol. 2001; 59: 386-392Crossref PubMed Scopus (197) Google Scholar; Fig.1). cytochrome P450 reverse transcription Recently we have shown that expression of members of another group of cytochrome P450s, the CYP2C, results, in addition to canonical mRNAs, in a variety of chimeric RNA molecules in human liver and epidermis (10Finta C. Zaphiropoulos P.G. Genomics. 2000; 63: 433-438Crossref PubMed Scopus (43) Google Scholar, 28Zaphiropoulos P.G. Nucleic Acids Res. 1999; 27: 2585-2590Crossref PubMed Scopus (38) Google Scholar). Here we provide evidence that chimeric RNA production is not limited to the CYP2C genes on chromosome 10q24, but is also typical of the CYP3A genes. Specifically, in liver several hybrid mRNAs are produced that encompass the first exon of CYP3A43 joined to various sets of eitherCYP3A4 or CYP3A5 exons. In addition, because theCYP3A43 gene has an opposite orientation to theCYP3A4 and CYP3A5 genes, the mechanism of chimeric mRNA formation presumably involves splicing events between CYP3A43 and either CYP3A4 or CYP3A5 pre-mRNA molecules. Thus, these findings suggest that natural trans-splicing between distinct genes may occur in higher eukaryotes. Moreover, this intricate pattern of expressed mRNAs implies that novel alternative splicing pathways may significantly increase the complexity of human gene expression. Human total RNA was purchased from Invitrogen (adult human liver) or purified from HepG2 cells. HepG2 cells (ATCC) were grown at 37 °C, in an atmosphere of humidified air containing 5% CO2, in RPMI 1640 with l-glutamine medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Subculturing was performed at a subcultivation ratio of 1:6. Cells from confluent cultures were harvested by addition of trypsin, and subjected to RNA preparation using the SV Total RNA Isolation System (Promega). cDNA synthesis was carried out with Moloney murine leukemia virus reverse transcriptase (Promega) in a 50-μl reaction mixture using oligo(dT) primers (25 ng/ml, Promega) with 10 μg of total RNA template. An aliquot of 1–5 μl of the reverse transcription reaction was directly subjected to the first amplification. Both the initial and nested polymerase chain reactions (PCRs) were performed in a 50-μl reaction mixture containing 20 pmol of each of the forward and reverse primers (Cybergene AB), 2 mm MgCl2, 200 μm dNTPs, and 1.25 units of Taq DNA polymerase (Promega). The oligonucleotides used in the PCRs are listed in Table I. The reactions were carried out for 30 cycles, with 10 s at 92 °C, 30 s at 52 °C, and 2 min at 72 °C. For the nested reaction, 1 μl of the first PCR was directly used. Alternatively, amplifications were done by using the Expand Long Template PCR System (Roche Molecular Biochemicals) for 30 cycles with 10 s at 92 °C, 30 s at 52 °C, and 2 min +5 s/cycle at 68 °C.Table IPCR primers and TaqMan probesPCR primersCYP3A4 exon 1, forward5′-AAA GAG CAA CAC AGA GCT GCYP3A4 exon 1, forward, nested5′-TCA GAG CAG AGA GAT AAG TCYP3A4 exon 13 reverse5′-GTC CTT AGG AAA ATT CAG GCYP3A4 exon 13 reverse, nested5′-TGC CAT CCC TTG ACT CACYP3A5 exon 1, forward5′-AAA CAG CAG CAC TCA GCT ACYP3A5 exon 1, forward, nested5′-TCA CAG AAC ACA GTT GAA GCYP3A5 exon 13, reverse5′-AGT CCT TAG AAT AAC TCA TTCYP3A5 exon 13, reverse, nested5′-GTT CCA TCT CTT GAA TCCCYP3A7 exon 1, forward5′-GCA GCA CGC TGC TGA AAACYP3A7 exon 1, forward, nested5′-TCA TCC CAA ACT TGG CCGCYP3A7 exon 13, reverse5′-CAT TTC AGG GTT CTA TTT GTCYP3A7 exon 13, reverse, nested5′-AGC TTT CTT AAA GAG CAA ACCYP3A43 exon 1, forward5′-ACA GAG CTG AAA AAG AAA ACCYP3A43 exon 1, forward, nested5′-TTG CCA TGG AAA CAT GGGCYP3A43 exon 13, reverse5′-CAA AAC AAT TTG AAG TGT CTACYP3A43 exon 13, reverse, nested5′-TGG TTG AAG AAT TGG TAG ATReal-time PCR primers and probesCYP3A43 exon 1, sense5′-CCA AAC TTT GCC ATG GAA ACACYP3A43 exon 2, antisense5′-GTT TAT GTG AAT GGG TCC CAT AAA TCYP3A43 TaqMan probe5′-6-FAM-TCT TGT GGC TAC CAG CCT GGT ACT CCT CT-TAMRACYP3A43/3A4 exon 1, sense5′-CAT GGG TTC TTG TGG CTA CCACYP3A43/3A4 exon 4, antisense5′-CAG GAT CTG TGA TAG CCA GCA CCYP3A43/3A4 TaqMan probe5′-TET-TGG TAC TCC TCT ATA TCT TTT ATG ATG GTC AAC AGC C-TAMRA Open table in a new tab The PCR products were cloned inEscherichia coli XL1-Blue cells using the pGEM-T Vector System (Promega). The cloned PCR products were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Electrophoresis of the sequenced samples was performed by Cybergene AB or KISeq (Karolinska Institute). The analysis of the DNA sequences was done with the BLAST similarity search program (29Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60532) Google Scholar). RNA was isolated from a human liver sample (a generous gift of Dr. Nikos Papadogiannakis, Huddinge University Hospital) by the guanidinium thiocyanate method (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63290) Google Scholar). A DNA construct containing a segment of the (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(2-3-4-5-6-7-8-9-10-11-12-13)3A4cDNA spanning 43 base pairs (bp) upstream and 25 bp downstream of the nontypical splice junction was generated by PCR amplification (10 cycles) of that cDNA with Advantage HF polymerase (CLONTECH) and primers 5′-GCG CGG CCG CAA ACA TGG GTT CTT GTG GC and 5′-GCG TCG ACA AGT CCA TGT GAA TGG GTT CCA TAT (Cybergene AB). The presence of NotI and SalI sites in the primers allowed the directional cloning of the PCR product into the pGEM-5Z (Promega) vector, and the sequence of the construct was confirmed by direct dye-deoxy sequencing. The construct was linearized with NotI (Promega) and transcribed (MAXIscript™, Ambion) in the presence of 32P-labeled UTP (Amersham Biosciences, Inc.) with either SP6 polymerase to generate the riboprobe or with T7 polymerase to produce a molecular weight marker for the analysis of the protected fragments. 50 and 150 μg of total RNA from human liver were incubated with equal amounts (2 × 105 cpm) of the riboprobe and subjected to RNase protection following the protocol of the RPA III™ kit (Ambion). Electrophoretic analysis of the riboprobe and the protected fragments was performed on a denaturing 10% acrylamide, 8 m urea gel and visualized after exposure to Fuji Super RX film. Aliquots (10 μg) of human genomic DNA (Promega) were digested individually with BglII,EcoRI, NdeI, and SpeI endonucleases (Promega). Following electrophoretic separation in a 0.8% agarose gel, DNA fragments were blotted to HyBond N membrane (Amersham Biosciences, Inc.) according to the supplier's recommendations. The DNA blot was subjected to Southern hybridization using the CYP3A43 exon 1-specific oligonucleotide, 5′-TAC CAG GCT GGT AGC CAC AAG AAC CCA TG (Cybergene AB), end-labeled with adenosine 5′-[γ-32P]triphosphate (>5000 Ci/mmol, Amersham Biosciences, Inc.). After hybridization using ExpressHyb hybridization solution (CLONTECH), the membrane was washed twice for 30 min at room temperature with 2× SSC, 0.1% SDS. This was followed by consecutive washing steps at gradually increasing stringent conditions, two washes for 30 min each at 55 °C with 2×, 1×, and 0.5× SSC. Radioactive signal was visualized either by autoradiography or by using a phosphorimaging analyzer (Fujix BAS2000). cDNA synthesis was carried out with Moloney murine leukemia virus reverse transcriptase (Promega) in a 13-μl reaction mixture using 20 pmol of consensus CYP3A exon 4-specific antisense primer, 5′-TAA CAT TCT TTC ACT A (Cybergene AB), with 15 μg of human liver total RNA template (Invitrogen). A 2-μl aliquot of the reverse transcription reaction was directly subjected to amplification. Quantitative real-time PCR (31Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5063) Google Scholar) was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) with an Applied Biosystems Sequence Detector 7700. The amplification reaction was carried out for 40 cycles, with 15 s at 95 °C and 1 min at 60 °C in a volume of 30 μl containing 200 nm PCR primers and 200 or 225 nmfluorogenic probe for the canonical CYP3A43 cDNA or the CYP3A43/3A4 cDNA containing exon combination (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(4-5-6-7-8-9-10-11-12-13)3A4, respectively. Primers and probes (Cybergene AB) are listed in Table I. To create calibration curves, known amounts of plasmids (1 pg, 100 fg, 10 fg, 1 fg, and 100 ag) encompassing CYP3A43 and (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(4-5-6-7-8-9-10-11-12-13)3A4CYP3A43/3A4 cDNAs were used, respectively. For cloning purposes, a 1-μl aliquot of the PCR reaction was subjected to a 10 cycle reamplification with 10 s at 92 °C, 30 s at 60 °C, and 1 min at 72 °C. The PCR was performed in 50 μl using 2.5 units of Taq polymerase with 2 mm MgCl2, 200 μm dNTPs, and 120 nm of each primer. Polyadenylated RNA was isolated by subjecting 120 μg of human liver total RNA (Invitrogen) to a two round purification with Poly(A) Spin mRNA Isolation Kit (New England Biolabs). Poly(A)+ RNA and flow-through were precipitated with ethanol and reverse-transcribed as described for total RNA (above). Two μl of the RT reaction were directly used in quantitative real-time PCR. CYP3A4 and chimeric proteins encoded by (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(2-3-4-5-6-7-8-9-10-11-12-13)3A4 and (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(4-5-6-7-8-9-10-11-12-13)3A4 RNAs were expressed under the control of human cytomegalovirus promoter in expression vector pcDNA1.1 (Invitrogen). CYP3A4 cDNA was isolated by RT-PCR on human liver RNA using the Advantage High Fidelity PCR system (CLONTECH) with CYP3A4 exon 1, forward, nested, and CYP3A4 exon 13 reverse primers (Table I). The RT-PCR product was cloned using the pGEM-T Vector System (Promega), sequenced, and subcloned on an SphI-SpeI fragment intoSphI-XbaI-digested pcDNA1.1 (Invitrogen). cDNAs of (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(2-3-4-5-6-7-8-9-10-11-12-13)3A4 and (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(4-5-6-7-8-9-10-11-12-13)3A4 trans-spliced species were isolated using 5′-GCG CGG CCG CGA TGG ATC TCA TTC CAA ACT TT forward (Cybergene AB) and CYP3A4 exon 13 reverse primers (Table I). In similarity with CYP3A4, CYP3A43/3A4 cDNAs were cloned using the pGEM-T System (Promega), sequenced, and subsequently cloned onSphI-SpeI fragments intoSphI-XbaI-digested pcDNA1.1 (Invitrogen). COS-7 cells (a generous gift of Cecilia Kut, Södertörns University College, Huddinge, Sweden) were grown at 37 °C, in an atmosphere of humidified air containing 5% CO2, in Dulbecco's modified Eagle's medium (with 1000 mg/liter glucose; Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and occasionally with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml gentamycin (Invitrogen). Subculturing was performed at a subcultivation ratio of 1:5. Cells were transfected using the DEAE-dextran method (32Gulick T. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997: 9.2.1-9.2.10Google Scholar) or using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. After 60–65 h of expression, cells were harvested in 0.01 m phosphate-buffered saline, pH 7.4, and cell homogenates were prepared by sonication and subsequent centrifugation (10,000 × g, 10 min). Enzyme activity of total COS-7 cell lysates and human liver microsomes (Biopredic) was measured for 60 min at 37 °C in 500 μl of 0.01 mphosphate-buffered saline, pH 7.4, 0.1 mm EDTA using 100 μm [4-14C]testosterone (Amersham Biosciences, Inc.) as substrate. The reaction was initiated by the addition of 1 mm NADPH (Sigma) and terminated by extraction with ethyl acetate. After extraction, separation and evaporation of the organic phase, products were dissolved in methanol and subjected for silica gel thin layer chromatography (J.T. Baker) in dichloromethane:acetone (3:1). Radioactive products were visualized using autoradiography. Systematic analysis of cytochrome P450 2C transcripts in human epidermis and liver revealed that, in addition to the canonical CYP2C mRNAs, several chimeric molecules were also produced (10Finta C. Zaphiropoulos P.G. Genomics. 2000; 63: 433-438Crossref PubMed Scopus (43) Google Scholar,28Zaphiropoulos P.G. Nucleic Acids Res. 1999; 27: 2585-2590Crossref PubMed Scopus (38) Google Scholar). These RNAs were characterized by encompassing exons from differentCYP2C genes, and some also had the potential to encode for chimeric CYP2C proteins. To test the hypothesis that intergenic mRNA formation is not only restricted to the CYP2Cgenes, we examined whether hybrid mRNA molecules were also formed between distinct members of the CYP3A gene cluster. Specifically, we performed nested RT-PCR on human liver RNA with primers from CYP3A43 exon 1 and CYP3A4 exon 13. The amplification resulted in three DNA fragments. Cloning and DNA sequence analysis revealed that these encompassed CYP3A43exon 1 spliced to CYP3A4 exons (Fig.2). The exons were joined at canonical splice sites, suggesting that the hybrid mRNA molecules were formed during pre-mRNA splicing. One combination consisted of all 13 exons characteristic of the CYP3A mRNAs and thus is capable of encoding for a chimeric CYP3A protein. Worth noting is that, in the other two combinations, the CYP3A4 exons that are spliced immediately after the CYP3A43 exon 1, i.e. exon 4 and exon 7, are in the same phase as exon 2. Therefore, all three detected mRNA molecules are characterized by open reading frames that start at the canonical ATG initiation codon and terminate at the canonical TGA termination codon (TableII).Table IICorrelation between translational reading frame and trans-splicingExonReading frameIntergenic species2−1+30−4−1+50−60−7−1+8+1−90−10+1−110+12−1+130−Note that all but one of the CYP3A exons that were found to juxtapose to the CYP3A43 exon 1 have a reading frame that is identical to the reading frame of exon 2. Open table in a new tab Note that all but one of the CYP3A exons that were found to juxtapose to the CYP3A43 exon 1 have a reading frame that is identical to the reading frame of exon 2. Nested RT-PCR amplification using 3A4 exon 1 forward and3A43 exon 13 reverse primers resulted in no products (data not shown). This suggests directionality in the formation of CYP3A43/3A4 intergenic mRNAs. To obtain additional evidence by a non-PCR-based method for the presence of intergenic CYP3A mRNAs in human liver, the RNase protection analysis was employed. Specifically, a riboprobe was generated (see "Materials and Methods") that spans 68 bases of the CYP3A43 exon 1 to CYP3A4 exon 2 splice junction. The CYP3A43 exon 1 overlap was chosen to be of sufficient length (43 bases) that would allow efficient hybridization under the conditions used; however, the CYP3A4 exon 2 overlap was minimized to only 25 bases, to destabilize formation of RNA duplexes with the highly abundant CYP3A4 mRNA. As anticipated, protected fragments of a size of 68 and 43 bases were clearly visible (Fig. 3). Moreover, the intensity of the protected fragments increased with increasing amounts of input human liver RNA. The ratio of the two protected fragments indicated that the joining of CYP3A43 exon 1 to CYP3A4 exon 2 is a much more rare event than the canonical joining of CYP3A43 exons 1 and 2. In similarity with the CYP3A43/3A4 chimeric mRNA molecules, CYP3A transcripts encompassing CYP3A43 and CYP3A5 exons are also formed in human liver. Specifically, nested RT-PCR amplification using 3A43 exon 1 forward and 3A5exon 13 reverse primers resulted in two fragments. Cloning and subsequent sequence analysis revealed that both consisted ofCYP3A43 exon 1 joined to CYP3A5 exons (Fig.4). All exons were spliced at canonical sites, indicating that CYP3A43/3A5 hybrid mRNA formation was a result of a pre-mRNA splicing process. The CYP3A43/3A5 chimeric mRNAs also have directionality, as no products were detected in a nested RT-PCR experiment with CYP3A5 exon 1 sense primers combined with CYP3A43 exon 13 antisense primers (data not shown). In contrast to the CYP3A43/3A4 and CYP3A43/3A5 intergenic mRNA molecules, no transcripts encompassing combinations betweenCYP3A43 and CYP3A7 exons could be detected. Nested RT-PCR amplification using 3A43 exon 1 forward and3A7 exon 13 reverse primers resulted in no product in human liver RNA (data not shown). Moreover, no product was detected with3A7 exon 1 sense and 3A43 exon 13 antisense primers either (data not shown). We also repeated the RT-PCR amplification using RNA isolated from the HepG2 hepatoblastoma cell line that is characterized by higher CYP3A7 expression than adult liver. However, RT-PCR amplification with 3A43 exon 1 sense and 3A7 exon 13 antisense primers still resulted in no product (data not shown). An interpretation of the mechanism that generates CYP3A43/3A4 and CYP3A43/3A5 intergenic mRNAs could be the existence of a duplicated CYP3A43 exon 1 sequence, having the same orientation as the CYP3A4 and the CYP3A5 genes, that is used in a cis-splicing process. To test this possibility, Southern hybridization was performed on human DNA digested with various restriction endonucleases. A CYP3A43 exon 1-specific oligonucleotide was used as the probe. Fig.5 shows that hybridization resulted in single bands in all four DNA samples. Moreover, the estimated sizes for the DNA fragments fit well with those deduced from the genomic sequence, i.e. 4240, 1224, 4928, and 1961 bp forSpeI-, NdeI-, EcoRI-, andBglII-digested DNA, respectively. These results provide strong evidence that there is only a single CYP3A43 exon 1 in the human genome and thus indicate that the CYP3A43 gene is indeed the source of the 3A43 exon 1 sequences in the CYP3A43/3A4 and CYP3A43/3A5 intergenic mRNA molecules. The absence of a duplicated CYP3A43 exon 1 is also supported by the available genomic GenBank™ entries that cover the CYP3A locus (Fig.1). Therefore, because the CYP3A43 gene is encoded by a different DNA strand than the other members of the CYP3Acluster, including the genes for CYP3A4 and CYP3A5, the formation of the identified intergenic CYP3A transcripts can only be rationalized by splicing between distinct pre-mRNA molecules, i.e. trans-splicing. To obtain a quantitative measurement of the abundance of the trans-spliced CYP3A mRNAs, the hybrid mRNA molecule containing exon combination (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (749) Google Scholar)3A43-(4-5-6-7-8-9-10-11-12-13)3A4, was analyzed by real-time 5′ exonuclease (TaqMan) PCR. Aliquots of human liver cDNA were subjected to quantitative PCR to measure the absolute amount of the canonical CYP3A43 cDNA and the (1Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Cell. 1991; 64: 607-61
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