Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Institute, Huddinge, Sweden
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Abstract |
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Introduction |
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Pre-mRNAs, the primary transcripts in eukaryotic cells, undergo a number of posttranscriptional modifications, including the excision of noncoding intronic sequences by large protein/RNA complexes called spliceosomes (Smith and Valcárcel 2000
). Conserved sequences necessary for the identification of exon/intron boundaries include two intronic dinucleotides, in most cases GT and AG, located, respectively, at the 5' and 3' splice sites (Burset, Seledtsov, and Solovyev 2000
; Smith and Valcárcel 2000
). A branch site sequence containing a conserved A nucleotide located close to the 3' splice site is also required for exon/intron recognition (Smith and Valcárcel 2000
).
There is increasing evidence that pre-mRNA splicing cannot be explained by the simple mechanism of the spliceosome scanning 5' to 3', removing introns, and joining exons in the order they are found in the primary transcript. The existence of low-abundant, nontypical mRNA molecules has been documented in numerous studies (reviewed in Finta and Zaphiropoulos 2000c
). RNA species containing scrambled exons, where consensus splice sites have been used to join exons in an order different from that of the genomic DNA (Nigro et al. 1991
; Cocquerelle et al. 1992
; Caldas et al. 1998
; Chao et al. 1998
; Marcucci et al. 1998
; Crawford et al. 1999
; Surono et al. 1999
; Takahara et al. 2000
), and exon repetition in the form of tandem repeats of exons which are not the result of gene duplications (Caudevilla et al. 1998
; Akopian et al. 1999
; Frantz et al. 1999
; Finta and Zaphiropoulos 2000a
) are examples of phenomena that indicate that pre-mRNA splicing is indeed a highly complex process. In addition, evidence of the existence of RNA molecules possessing properties of circular molecules has been presented (Capel et al. 1993
; Cocquerelle et al. 1993
; Pasman, Been, and Garcia-Blanco 1996
; Li and Lytton 1999
). Furthermore, nontypical splicing events may result in the joining of canonical exons with sequences, both exonic and intronic, from pseudogenes or neighboring genes belonging to the same gene family (Benson, Nguyen, and Maas 1995
; Zaphiropoulos 1999
; Finta and Zaphiropoulos 2000b
), as well as unrelated nearby sequences (Shimamura et al. 1998
; Rogalla et al. 2000
).
The present study was directed at investigating splice variants within the human CYP2C subfamily, specifically species encompassing a novel CYP2C18 exon 1like sequence. Moreover, with the use of six independent bacterial artificial chromosome (BAC) clones, the gene locus containing the CYP2C18 exon 1like sequence was characterized.
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Materials and Methods |
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Isolation of BAC DNA
BAC clone RP11-400G3 was obtained from BACPAC Resources. BAC clones CIT-342L6, RP11-622P11, CIT-2061B9, CIT-2339N22, and CIT-3052K18 were supplied by Research Genetics. BAC DNA for PCR analysis of the six BAC clones for the presence of CYP2C19 exon 9, CYP2C9 exon 1, and the CYP2C18 exon 1like sequence was isolated using the JetQuick Plasmid Miniprep kit (Genomed), but with phenol/chloroform extractions and an ethanol precipitation instead of column purification. BAC DNA for the Southern blot was obtained using the Concert High Purity Plasmid Maxiprep System (Life Technologies) with an isopropanol precipitation included before column loading.
PCR Amplifications
PCR amplifications were performed using either the Perkin Elmer 480 thermal cycler or the MJ Research PTC-200 DNA Engine. All reactions were carried out for 30 cycles, unless stated otherwise, in a total volume of 50 µl using the hot start method. For the nested rapid amplification of cDNA ends (RACE) and intergenic PCR amplifications, 1 µl of the initial amplification products was used. Gene-specific primers (table 1
) were synthesized by Cybergene AB (Huddinge, Sweden).
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Intergenic PCR
For the set of experiments performed to identify intergenic products containing the CYP2C18 exon 1like sequence, 1 µl human liver cDNA synthesized as described above was the template in all cases. The reaction mixture for amplifications using Taq DNA polymerase (Promega) contained Taq buffer, 2 mM MgCl2, 200 µM dNTPs, 90 pmol of each primer (table 1
), and 5 U Taq polymerase. With Expand polymerase, the reaction mixture was as described above. For the Perkin Elmer 480 thermal cycler, denaturation was at 94°C for 1 min, annealing at the appropriate temperature (14°C below the calculated melting temperature) for 1 min, and extension at 72°C for 13 min (+4 s/cycle). When using the MJ Research PTC-200 DNA Engine, amplifications were performed with 10 s at 92°C, 30 s at the appropriate annealing temperature, and 2 min at 72°C.
Genomic PCR
The BAC clones were characterized by PCR using Taq DNA polymerase essentially as described above. Denaturation was at 92°C for 10 s, annealing at the appropriate temperature for 20 s, and extension at 72°C for either 40 s or 13 min (+4 s/cycle). Longer fragments of the RP11-400G3 BAC clone were amplified using the Expand Long Template PCR System with Expand buffer 3, 500 µM dNTPs, 20 pmol of each primer (table 1 ), and 3.5 U of Expand enzyme. The reactions were carried out with denaturation at 92°C for 10 s and annealing at the appropriate temperature for 20 s. The extension step was performed for 510 cycles of 10 min at 68°C, followed by 1520 cycles at the same temperature for 10 min + 20 s/cycle.
Cloning and Sequencing
The PCR products from the RACE experiments were purified directly using the JetQuick Gel Extraction Spin Kit (Genomed), digested with the restriction enzymes NotI and SalI, and ligated to a pGEM-5 vector (Promega). PCR products from all other experiments were directly cloned in a pGEM-T vector (Promega). Electrocompetent Escherichia coli XL1-Blue cells were transformed and clones containing plasmids with inserts were identified using blue/white selection. Plasmid DNA was isolated from overnight cultures of selected clones using the Jetquick Plasmid Miniprep Spin Kit (Genomed). Analysis of PCR products and plasmid DNA was carried out on 0.7%2% agarose gels. Sequencing of clones was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Electrophoresis of the sequenced samples was carried out at KISeq (Centre for Genomics Research, Karolinska Institute, Solna, Sweden) or Cybergene AB (Huddinge, Sweden). DNA sequences were analyzed using the NCBI BLAST similarity search tool (Altschul et al. 1997
).
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Results |
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3' RACE Amplifications of the CYP2C18 Exon 1like Sequence
With the purpose of acquiring more information concerning the expression of the CYP2C18 exon 1like sequence, 3' RACE amplifications were performed on human liver cDNA using forward primers specific for the CYP2C18 exon 1like sequence. Cloning and sequencing of the products from the 3' RACE experiments resulted in the detection of five species (fig. 2A
). Three splice variants were identified, with the CYP2C18 exon 1like sequence spliced either to sequences from the CYP2C9 gene or to a sequence originating from the CYP2C locus. In all cases, splicing had occurred via recognition of the canonical GT-AG dinucleotides. This provides evidence that the PCR products analyzed represent preexisting RNA molecules and do not originate from in vitro recombination events during PCR (Zaphiropoulos 1998
). The two additional species were found to comprise the CYP2C18 exon 1like sequence followed by contiguous intronic sequences. The results of the 3' RACE experiments would seem to indicate that the CYP2C18 exon 1like sequence is used for splicing only to sequences from the CYP2C9 gene and not to other members of the gene cluster.
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The CYP2C18 Exon 1like Sequence Is Positioned in the CYP2C19/CYP2C9 Intergenic Region
The GenBank entry AL133513 corresponding to the BAC clone RP11-400G3 contains, in addition to the CYP2C18 exon 1like sequence, exons 69 of CYP2C19 and exons 15 of CYP2C9. In order to investigate whether the BAC clone encompasses any further sequences from the CYP2C19 and CYP2C9 genes, efforts were undertaken to amplify exon 1 of CYP2C19 and exon 9 of CYP2C9 from the BAC clone. These experiments did not result in the detection of any PCR product, implying that the BAC clone contains only the 3' end of CYP2C19 and the 5' end of CYP2C9.
In order to provide evidence for the exact positioning of the CYP2C18 exon 1like sequence relative to the 3' end of CYP2C19 and the 5' end of CYP2C9, additional BAC clones containing sequences from this region of the CYP2C locus were identified using the TIGR Human BAC Ends database (Institute for Genomic Research, http://www.tigr.org). Five BAC clones were selected and analyzed for the presence of exon 9 of CYP2C19 and exons 1 of CYP2C9 and CYP2C18-like (fig. 3 ). These results, taken together with the finding of a contiguous sequence containing CYP2C9 exons 15 in two independent GenBank entries (AL133513 and AL359672), provide unambiguous evidence that the CYP2C18 exon 1like sequence is located in the CYP2C19/CYP2C9 intergenic region.
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Attempts were also made to link the CYP2C18 exon 1like sequence to the CYP2C9 and CYP2C19 genes by carrying out PCR amplifications of the BAC clone RP11-400G3. The use of a CYP2C18 exon 1like forward primer in combination with a CYP2C9 exon 1 reverse primer did not result in any product. In parallel with this experiment, amplification of exons 15 of CYP2C9 was used as a positive control and produced the expected 10.5-kb species. A second amplification using a CYP2C18 exon 1like reverse primer together with a CYP2C19 exon 9 forward primer was performed in order to try to connect the exon 1like sequence to the CYP2C19 gene. This experiment also failed to detect the desired species, implying an extensive CYP2C19/CYP2C9 intergenic region, hence the failure of long-range PCR to detect products. These findings, together with the results of the analysis of the six BAC clones, support the most recent update of the AL133513 entry (version of March 20, 2001; GI: 13396316), which reveals an 86-kb CYP2C19/CYP2C9 intergenic region with the CYP2C18 exon 1like sequence located 33 kb upstream of CYP2C9. Furthermore, this update provides evidence that the sequence from the CYP2C locus identified in one of the RACE splice variants (fig. 2A ) originates from the CYP2C18 exon 1like/CYP2C9 intergenic region.
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Discussion |
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The discovery that exon-like sequences located in intergenic regions do not necessarily represent inactive pseudogenes has previously been reported in the case of the CYP3A7 gene in chromosome 7q21q22.1. An alternatively spliced CYP3A7 mRNA form was detected where two "pseudogene" exons provide the coding information for a variant carboxyl terminus (Finta and Zaphiropoulos 2000b
). However, in the present report, the CYP2C18 exon 1like sequence is under the control of its own promoter and is expressed in combination with exons from the neighboring CYP2C9 gene. Interestingly, even sequences from the 5' flanking region of the CYP2C9 gene have been captured as spliced exons in transcripts originating from the promoter of the CYP2C18 exon 1like sequence.
These findings interpreted from the perspective of the CYP2C9 gene may also be viewed as follows: CYP2C9 transcription does not necessarily start between the putative TATA box (de Morais et al. 1993
) and the translation start site. Instead, some transcripts may initiate at promoter sequences upstream of the coding segment of the CYP2C18 exon 1like sequence. The ATA box (fig. 1
) detectable at the position equivalent to the TATA boxes of the CYP2C18, CYP2C9, and CYP2C19 genes may allow transcription, albeit at a lower efficiency than that of the canonical CYP2C18 gene. The CYP2C9 gene might therefore be considered part of a complex transcription unit having more than one promoter and undergoing several alternative splicing events.
Significance of the Transcripts Produced by the CYP2C18-like Promoter
Is there a coding potential in the transcripts generated by the CYP2C18-like promoter? Well, most transcripts contain termination signals in frame with the open reading frame of the exon 1like sequence. Moreover, no transcript was identified where the canonical exon 1 of CYP2C9 had been replaced with the CYP2C18 exon 1like sequence, resulting in a typical CYP2C mRNA of nine coding exons, even though CYP2C intergenic transcripts corresponding to a standard-length CYP2C mRNA have been observed in a previous study (Finta and Zaphiropoulos 2000a
). The transcript composed of exons 69 of CYP2C9 spliced after the exon 1like sequence (fig. 2B
) does, however, have an open reading frame that starts at the Met codon of the exon 1like sequence and ends at the canonical TGA codon of CYP2C9. Moreover, the additional 5' sequence present in the transcript containing the nine typical CYP2C9 exons (fig. 2B
) may have a role in modulating the translational ability of that mRNA (Morris and Geballe 2000
).
The results of the RACE and PCR amplifications clearly indicate that transcription is ongoing at the CYP2C18 exon 1like locus. The fact that these transcripts are not very abundant may relate to the observation that they do not represent typical CYP2C mRNAs of nine exons. These transcripts are, however, processed RNA molecules; that is, they have undergone splicing events, thereby discriminating them from unprocessed transcripts. Examples of the latter are species originating from the CYP3AP1, the CYP3AP2 (Finta and Zaphiropoulos 2000b
), and the CYP3A43P (GenBank entry AC011904, GI: 8051573, nucleotide positions 113048112978) (unpublished data) pseudogenes. These transcripts contain only exon-like sequences with the contiguous unspliced intronic sequences. We therefore favor the possibility that the CYP2C18 exon 1like transcripts represent efforts of the transcriptional/splicing machinery to generate exon combinations that may be useful to the cell (Finta and Zaphiropoulos 2001
). This would be in line with genomewide analyses of transcriptome sequences that suggest the presence of a large number of transcripts deviating from typical mRNAs (Velculescu et al. 1999
; Croft et al. 2000
; Hirosawa et al. 2000
). However, at this point in evolutionary time, no single CYP2C18-like/CYP2C9 combination predominates, as is the case for the CYP3A7 carboxyl terminus variant (Finta and Zaphiropoulos 2000b
). On the other hand, these sequences have the potential to generate functional products, as the cell is continuously experimenting with various possibilities that may result in a "winning" combination.
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Acknowledgements |
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Footnotes |
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1 Keywords: pseudogene
alternative promoter
pre-mRNA splicing
gene evolution
Homo sapiens
cytochrome P450
2 Address for correspondence and reprints: Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Institute, SE-141 57 Huddinge, Sweden. peter.zaphiropoulos{at}cnt.ki.se
.
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