The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference
© Piehler et al; licensee BioMed Central Ltd. 2008
Received: 17 December 2007
Accepted: 11 April 2008
Published: 11 April 2008
Pseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (ABCA17P). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents.
Systematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene ABCC6, ABCC6P1 and ABCC6P2, are transcribed. ABCC6P1 and ABCC6 possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene ABCC6P1 resulted in a significant reduction of ABCC6 mRNA expression levels.
The human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of ABCC6 mRNA levels by targeted suppression of the expression of its pseudogene ABCC6P1 provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.
Human ATP-binding cassette (ABC) transporters form a large family of structurally highly related membrane transport proteins. Based on sequence similarities, the human ABC transporter family is categorized into seven subfamilies, denoted ABC A-G, and consists of 48 members. ABC transporters translocate a wide range of substrates across cellular membranes, e.g. lipids, sugars, peptides, ions and xenobiotics such as anticancer drugs [1, 2]. Defective ABC transporters cause monogenetic disorders and contribute to the development of complex diseases . The physiological importance of ABC proteins is highlighted by the fact that mutations in 14 human ABC transporter genes have thus far been linked to a spectrum of unrelated hereditary diseases .
Beside protein coding genes for ABC transporters, it has been shown that the human genome contains several ABC transporter pseudogenes . However, the exact number of ABC transporter pseudogenes present in the human genome is still unclear. Generally, pseudogenes are defined as non-functional sequences that are present in the genome with high similarity to one or more paralogous genes. This lack of function is a result of either failure of transcription or translation, or production of a protein that differs in function from the protein encoded by the functional gene . Pseudogenes are thought to arise either by gene duplication and subsequent degeneration of the duplicated gene or by retroposition of a reversely transcribed mRNA of the paralogous gene into the genome. The latter are intronless and referred to as "processed" pseudogenes .
During the past years, we and others provided evidence for the existence of ABC transporter pseudogenes which are transcriptionally active [7–9]. For example, we recently demonstrated that the human ortholog of the rodent testis-specific ABC transporter Abca17, a protein coding gene, is a pseudogene (ABCA17P) which is transcribed into up to 20 alternative splice variants . These results raise the intriguing possibility that ABC transporter pseudogene transcripts may serve biological functions other than providing the genetic blueprints for protein biosynthesis.
In this study, we applied a comprehensive bioinformatic approach to identify all ABC transporter pseudogenes in the human genome and systematically searched for evidence of transcribed pseudogenes. We identified a total of 22 pseudogenes within the ABC transporter family with a significant subfraction (n = 10) exhibiting transcriptional activity. We find evidence for transcription of the two previously known ABCC6 pseudogenes ABCC6P1 and ABCC6P2, respectively, and report their gene structures. Moreover, we demonstrate, for the first time, a regulatory interdependency between ABCC6P1, and its translated counterpart, the pseudoxanthoma elasticum gene ABCC6.
The human genome contains 22 ABC transporter pseudogenes
ABC transporter pseudogenes in the human genome
Evidence for transcription
Alternatively spliced transcripts
A total of 10 ABC transporter pseudogenes (45%) were intronless, directly flanked by repetitive sequences (as identified with the RepeatMasker track, UCSC Genome Browser), and most of them (n = 8) also exhibited poly(A)-tails. These structural features are diagnostic of processed pseudogenes  strongly suggesting that they originated from retrotransposed transcripts of ancestral functional ABC transporter genes (Table 1).
A significant portion of human ABC transporter pseudogenes are transcriptionally active
Gene structures and expression profiling of ABCC6 and its pseudogenes ABCC6P1 and ABCC6P2 in various human tissues
We also found in silico evidence for transcription of the second ABCC6 pseudogene, ABCC6P2. Consistent with this, our RT-PCR experiments using pooled mRNA from 20 human tissues confirmed expression of this pseudogene (Figure 2). ABCC6P2 is composed of four exons whose lengths vary between 59–183 bp which exhibit highest sequence similarity (98–100%) with the corresponding exons of its parental gene. Intron sizes of ABCC6P2 range from 0.1 to 1.7 kb. Interestingly, Genbank mRNA database searches revealed a 0.7 kb poly(A)-tail transcript which displays perfect identity with ABCC6P2 exons 1 and 2 plus additional 504 bp of the second intron (Figure 3). This transcript has been independently identified in the Mammalian Gene Collection Program  from the kidney [GenBank: BC015978].
The striking co-expression of the ABCC6 and ABCC6P1 raises the possibility that transcription of both genes is controlled by similar regulatory elements. When we compared the 5' regulatory region sequences of both genes, we found that they share an overall sequence similarity of 98.9% within the first 2.5 kb upstream of the start codon, the region that contains the putative core promoters (Additional File 2). Of note, 68 out of the 72 potential transcription factor binding sites, which we identified within the predicted ABCC6P1 promoter region, are fully intact. In particular, an NF-κB consensus binding sequence (pos. -233 bp), known to confer high-level ABCC6 expression in HepG2 hepatoma cells , is completely conserved in the putative ABCC6P1 promoter region. Three binding sites, a glucocorticoid response element (GR, pos. -2116 bp), an octamer binding factor (oct-1, pos. -1746 bp) and a GATA-3 consensus site (pos. -1269 bp), respectively, show single base exchanges. In addition, a USF binding site (pos. -622 bp) exhibits a nonamer-deletion which disrupts the respective consensus sequence. Intriguingly, single base exchanges give rise to five novel transcription factor binding sites (SRY -2381 bp, C/EBPb -1223 bp, GATA-2 -1124 bp, AML-2 -464 bp, C/EBP -316 bp) in the predicted ABCC6P1 promoter.
Comparison of the ABCC6 promoter sequence with the ABCC6P2 5' UTR revealed an even higher degree of homology (99.6%) than that observed for ABCC6P1. A total of 71 out of 72 transcription factor binding sites were fully conserved strongly suggesting that the ABCC6P2 expression pattern is highly similar to that of ABCC6 and ABCC6P1, respectively. We were unable, however, to quantitate ABCC6P2 expression levels by real-time PCR because the small size of the transcript and its exceedingly high sequence homology with ABCC6 and ABCC6P1 hampered specific detection of ABCC6P2 mRNA.
Targeted knockdown of the pseudogene ABCC6P1 results in downregulation of ABCC6 mRNA expression
In our study, we report the existence of 22 human ABC transporter pseudogenes. The total number of pseudogenes in the human genome has been estimated to ~20000 [13–15], whereas the predicted number of protein coding genes amounts to 20000 – 25000 . This estimation indicates a genome-wide gene/pseudogene ratio of app. 1:1. Given the existence of 48 functional human ABC transporters, the ABC transporter genes/pseudogene ratio is app. 2:1. Thus, the number of ABC transporter pseudogenes in the human genome is significantly lower than the expected average frequency of pseudogenes within currently identified gene-pseudogene families [17–19].
Our results indicate that the pseudogenes of the ABC transporter family are distributed unevenly across the chromosomes. Approximately one-half of the ABC transporter pseudogenes (n = 11) is dispersed across several chromosomes, whereas the other half is clustered on chr. 15 and 16, respectively. Of these, seven pseudogenes are located on the short arm of chr. 16 within a segment of 30 Mb (16p11.2-p13.3) and three processed (intronless) pseudogenes are clustered on the long arm of chr. 15. The ABC transporter pseudogene cluster on chr. 16p consists of three protein-coding ABC transporter genes, six duplicated pseudogenes and one processed pseudogene. Clustering of pseudogenes has been observed for other gene families in various vertebrates including the group of cytochrome P450 enzymes, α-globin, and the variable gene segments of the immunoglobulin heavy and light genes, respectively [17, 20, 21]. These gene clusters are usually composed of both protein coding genes and pseudogenes from the same gene family, which likely reflects the fact that pseudogenization is a mechanism that is highly associated with gene duplication events. The close spatial proximity of genes and pseudogenes suggests that the gene duplications are more recent evolutionary events because dispersion across the genome has not yet occurred. In light of this, it is reasonable to assume that the ABC transporter pseudogenes located on chr. 15 and 16 are indeed the result of gene duplication and subsequent pseudogenization of the previously functional ABC transporter genes. Of note, the rodent genome contains protein-coding genes for Abca14, Abca15  and Abca17 . It is thus conceivable that the human orthologs of these intact genes were functional for a certain time period after duplication before they ultimately became disabled. As for ABCC6P1 and ABCC6P2, the other two duplicated pseudogenes on chr 16, it remains unclear whether they arose by complete duplication of the parental ABCC6 gene and thus may have temporarily maintained their function or whether only fragments of this gene were duplicated ab ovo.
Unlike in the case of non-processed pseudogenes, the chromosomal distribution of processed pseudogenes generally appears to be random with the number of pseudogenes per chromosome being proportional to chromosomal length . This is consistent with the view that processed pseudogenes originate from nondirected retrotransposition of preexisting transcripts. Interestingly, the pseudogene cluster on chr. 15 contains three processed pseudogenes (ABCB10P1-3) which are localized in close proximity to each other (their parental gene ABCB10 is located on chr. 1). Because it is unlikely that the ABCB10P cluster is the result of three independent retrotransposition events targeting one and the same chromosomal segment, two of the three ABCB10 pseudogenes most likely arose by gene duplication. The homology analyses, which we conducted, indeed suggest that ABCB10P1 may have originated from ABCB10 by a retrotransposition process. Intriguingly, they also strongly support the view that ABCB10P2 arose by duplication of ABCB10P1 and ABCB10P3 is the result of the duplication of ABCB10P2 (Additional File 3). Because ABCB10P2 and ABCB10P3 most likely arose by duplication of an already disabled processed pseudogene, it is reasonable to assume that these pseudogenes never had the capacity to encode proteins. To our knowledge, the ABCB10P cluster thus represents the first example of the multiplication of a processed pseudogene in the human genome.
Importantly, we obtained bioinformatic evidence indicating that app. one-half (45%) of the human ABC transporter pseudogenes are transcriptionally active. Consistent with this, our RT-PCR based survey demonstrated that at least 7 out of the predicted transcriptionally active pseudogenes were in fact transcribed. Little attention has so far been paid to the phenomenon of pseudogene transcription  owing to the fact that pseudogenes are generally thought to be functionless genomic relics. However, this view is challenged by some recent studies. For example, a comprehensive study focussing on transcription of processed pseudogenes in the human genome revealed that a subfraction of app. 5% of all processed pseudogenes identified are transcribed . Moreover, analysis of pseudogene transcription on human chr. 22 using a combined bioinformatic/microarray approach demonstrated that up to 20% of the pseudogenes identified on this chromosome are transcriptionally active . The surprisingly high proportion of potentially transcribed pseudogenes questions the traditional view of pseudogenes and raises the intriguing possibility that transcribed pseudogenes may serve defined biological functions in the genome. The concept is supported by two recent studies which provide experimental evidence for pseudogene function. Korneev et al. reported direct interference of a gene and its transcribed pseudogene by demonstrating that the antisense pseudogene anti-NOS, whose transcript forms stable RNA-RNA complexes with the mRNA of nNOS, inhibits protein expression of its parental gene . Another study highlights the fact that pseudogene expression can exert a biological function that is unrelated to its ancestral gene as evidenced for the human Xist noncoding RNA gene, which initiates X chromosome inactivation and has evolved in eutherians by pseudogenization of the protein coding gene Lnx3 .
To test whether and to which degree regulatory interdependencies exist between transcribed ABC transporter pseudogenes and their protein coding counterparts, we selected the pseudoxanthoma elasticum gene, ABCC6, and its transcribed pseudogene ABCC6P1. This gene-pseudogene pair appeared to be an ideal candidate for interference studies because ABCC6 and ABCC6P1 exhibit near perfect sequence homology in their transcripts but, at the same time, can still be discriminated by RT-PCR due to non-homology disparity in the final exon (exon 10) of ABCC6P1. Moreover, this sequence disparity allowed the selective targeting of the pseudogene transcript using an siRNA approach. mRNA expression profiling of ABCC6 and ABCC6P1 revealed that both genes are consistently co-expressed in a wide range of human tissues with predominant expression in adult liver, kidney and fetal liver (in this order). The tissue expression pattern we observed for ABCC6 is in keeping with a recent genome-wide gene expression study involving 79 human tissues . Regrettably, this work does not include expression data on the pseudogene ABCC6P1. Our finding that ABCC6 and ABCC6P1 share near-perfect identity in their respective core promoter regions together with the observed co-expression pattern strongly suggests that the expression of the gene-pseudogene pair is governed by the same transcription factor machinery.
Intriguingly, we found that selective knockdown of ABCC6P1 leads to a reduction of ABCC6 mRNA expression suggesting that the expression of ABCC6P1 and its parental gene are interdependent. At this point, the regulatory mechanisms which underlie the observed gene-pseudogene interference remain speculative. It is well established that mRNA stability and degradation significantly depends on specific cis-acting elements in mRNA molecules. These cis-acting destabilizing elements, which can be found both in the UTR and the coding regions of mRNA are attacked by trans-acting machineries facilitating mRNA decay . Transcripts from distinct genes which exhibit extensive sequence homology (as in the case of gene-pseudogene pairs) and thus contain identical destabilizing cis-elements may compete for such trans-acting elements. It is thus conceivable that the ABCC6 and the ABCC6P1 transcripts due to their high sequence similarity and their topological proximity compete for the same cellular RNA degradation route. As a consequence, lower abundance in ABCC6P1 transcripts may thus lead to an increased ABCC6 mRNA degradation rate. It will be challenging to characterize in more detail the molecular interdependency between the ABCC6 and ABCC6P1 transcription units. Knowledge of ABCC6/ABCC6P1 regulatory interaction may be of potential relevance to clinical medicine because ABCC6 is the underlying gene defect in the syndrome pseudoxanthoma elasticum.
In this study, we established the existence of 22 ABC transporter pseudogenes in the human genome. Combined bioinformatic and experimental results indicate that a significant portion of these pseudogenes (45%) are transcribed and we provide evidence, for the first time, for expression of alternatively spliced pseudogene RNAs in the human genome. Moreover, we show that ABCC6P1, one of the pseudogenes of the pseudoxanthoma elasticum gene ABCC6, is co-expressed with ABCC6 in a variety of tissues and that knockdown of ABCC6P1 expression impacts the expression of ABCC6 strongly suggesting a regulatory interdependence between ABCC6P1 and its ancestral gene. The emerging concept of pseudogene transcription and functionality has recently found its reflection in the redefinition of pseudogenes as "genomic sequences that arise from functional genes but that cannot encode the same type of functional product [...] as the original gene" . Accordingly, a subclassification into (i) 'ghost' pseudogenes with some intermediate functionality, i.e. similar to a functional gene and transcribed and/or functional, and (ii) 'dead' pseudogenes that lack any functionality has recently been proposed. Our results presented here strongly support this novel differentiated view on the biology of pseudogenes.
Bioinformatic identification of human ABC transporter pseudogenes
To identify all ABC transporter pseudogenes in the human genome, we retrieved the complete sequence of the human genome  chromosome-wise from the UCSC Genome Browser (assembly March 2006, NCBI Build 36, hg18) [32, 33].
A stand-alone version of the program MaskSeq was used to mask off the exons of all human reference ABC transporter genes. For this, a list of the reference nucleotide sequence accession numbers of all human ABC transporters was used to retrieve the exon coordinates of the human protein coding ABC transporter genes from the Table Browser application of the UCSC Genome Browser . A Perl script was designed to extract and reformat the exon coordinates from the Table browser output for application in the program MaskSeq.
In addition to all human reference ABC transporter sequences, we downloaded the reference nucleotide sequences of all known mouse ABC transporters that have no apparent orthologous counterparts in the human genome (Abca14, Abca15, Abca16, Abca17, Abcg3). Using a local installation of the BLAST application (version 2.2.14), a homology search of the reference ABC transporter nucleotide sequences was performed against the human genome masked for ABC transporter exons. In this alignment, an E-value of 0.001 and the table output format "-m8" was applied. To eliminate hits originating from repetitive sequences, the repetitive sequences in the query ABC transporter nucleotide sequences were identified by applying the program RepeatMasker (version 3.1.6) using the database Repbase (version 11.12). An in-house written Perl script was used to exclude all hits that were located completely within repetitive sequences and those that extended less than 30 bp into the flanking regions of repetitive sequences. The remaining hits were then ordered along the chromosomes and merged to a single candidate pseudogene when neighbouring hits yielded highest homology to the same protein coding gene and when they were in the same 5'→3' orientation as their homologous ABC transporter nucleotide sequence. In the absence of a neighbouring hit, a genomic region of 0.1 Mb flanking the single hit was aligned with the respective ABC transporter nucleotide sequence using the more rigorous alignment tool "matcher" from the EMBOSS package. This approach allows for identification of more distantly related pseudogenes. Hits displaying > 70% identity over an entire exon of the respective ABC transporter nucleotide sequence, were also considered homologous and further evaluated. Merged hits were classified as candidate pseudogenes when (i) the retrieved sequences showed homologies to at least two exons of a protein coding gene and (ii) the merged hit contained sequence aberrations precluding its translation into a functional ABC transporter. Subsequently, all potential pseudogenes were analyzed using the UCSC Genome Browser  to exclude the possibility that candidate pseudogenes were part of a known protein coding gene. Additionally, the human mRNA and EST track of the UCSC Genome Browser were used to search for transcription of the identified ABC transporter pseudogenes. A locus was regarded a candidate transcriptionally active pseudogene when a homologous mRNA or EST sequence exhibited overall > 99% with the pseudogene but no other sites in the human genome ("best hit").
Sequence similarities and multiple sequence alignments were calculated using EMBOSS package algorithms [35, 36]. Potential transcription factor binding sites were identified utilizing tfsearch (version 1.3, threshold score 85.0, classification: vertebrate)  and the TRANSFAC database (release 3.3) .
Confirmation of pseudogene transcription
Aliquots of 1 μg pooled total RNA (Human Total RNA Master Panel II, Clontech) were reverse-transcribed in the presence of oligo-dT primers in a 20 μl RT reaction using the Omniscript RT Kit (Qiagen). cDNA was amplified using the HotStarTaq DNA polymerase Kit (Qiagen). The cycling conditions were 94°C 30s, 54–60°C 30s and 72°C 30s-3 min followed by a final extension step at 72°C for 10 min. Transcription of pseudogenes was authenticated utilizing the following primer sets: ABCA10P 5'-GATTTTGCCAGAGCCTCAGC-3', 5'-GAGTGCATTTGCTGCTTGGA-3', ABCA15P2 5'-AACTGGCATGGACCCAGTAG-3', 5'-CTTCCTCCAAAATGCCAAAC-3', ABCA17P 5'-TCACGCACTGTCTTTCCTTG-3', 5'-CCAGTACAATGGAACTGATGATG-3', ABCC2P 5'-GCCTGGATTCAGAATTGCAT-3', 5'-GCCCCACAAGAAAGCATAAA-3', ABCC6P1 5'-CCCACGACGACAGAAGG-3', 5'-TTCGGAAACCTTGGCTCA-3', ABCC6P2 5'-CCCACGACGACAGAAGG-3', 5'-TCTGTTTGGGAGAACCGTGT-3' and ABCG2P2 5'-GGTACTCCGCTGACACCTTC-3', 5'-TTTCTCAACTGGTTTTTGACCA-3'. PCR products were separated and analyzed on GelStar® (Cambrex) stained 0.8 – 1.5% agarose gels. Bands of interest were excised and the DNA was extracted using the QIAquick® Gel Extraction Kit (Qiagen). PCR runs resulting in a single band were purified using Microcon® YM-30 columns (Millipore) for subsequent sequence analysis. Purified amplification products were sequenced as published previously . Sequence data were analyzed using the program FinchTV v.1.3.1 (Geospiza Inc.) and the UCSC Genome Browser.
HepG2 cells were purchased from ATCC (no. HB-8065) and cultivated under standard conditions (5% CO2, 37°C) in ATCC complete growth medium (Eagle's medium) with 2 mM L-glutamine and Earle's BSS (1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%) containing 2% penicillin/streptomycin.
siRNA mediated knockdown of ABCC6P1 in HepG2 cells
21 mer-siRNAs were designed using the siRNA design tool from the Whitehead Institute for Biomedical Research , which target the sequence of exon 10 of the pseudogene ABCC6P1. This exon displays no homology (sequence identity < 20%) with its parental gene ABCC6 and thus allows selective targeting of ABCC6P1. siRNAs including positive (Silencer GAPDH siRNA) and negative controls (Silencer Negative Control #1 siRNA) were purchased from Ambion, Inc. After screening of several siRNAs, the following siRNAs which yielded the highest knockdown rates of ABCC6P1 were used in all further experiments: Sense 5'-CUCCCAGGGAUAUAAAUCAUU-3', and antisense 5'- UGAUUUAUAUCCCUGGGAGUU-3', respectively. HepG2 cells were reverse-transfected using 1.5 μl siRNA (20 μM) complexed with 3 μl siPORT™ NeoFX™ transfection agent (Ambion) in 100 μl OPTI-MEM buffer (Invitrogen). After 10 min incubation, the siRNA/lipid-complex was mixed with 900 μl of 105cells/ml HepG2 cells in culture medium and plated in 12 well plates. After 24 h incubation under standard conditions the cells were lysed, RNA was extracted (MagNA Pure LC RNA Isolation Kit – High Performance, Roche) and quantitated on a Nano Drop® spectrophotometer. All siRNA experiments were performed in duplicate, and the results of six independent siRNA experiments with ABCC6P1 knockdown rates > 60% were used for statistical analyses.
For gene expression profiling of ABCC6 and ABCC6P1 in different human tissues, 1 μg total RNA from each tissue type of Clontech's Human Total RNA Master Panel II, and for quantitation of transcription levels in the siRNA experiments, 150–250 ng total RNA from HepG2 cells were reverse transcribed using Omniscript (Qiagen) and oligo-dT primers. For quantitation of the transcription levels of selected genes real-time PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using standard conditions. The following TaqMan-primer/probes (Applied Biosystems) were used: ABCC6P1 (designed by Applied Biosystems): forward primer 5'-CCATCACTGGCCTGGTGTA-3', reverse primer 5'-CTCCTGCTGAAATCATCTGAGATTAGT-3', FAM-reporter probe 5'-CTGTGGATGCCTTTCTG-3' (targeting exon boundary 9/10 of ABCC6P1), ABCC6 (Assay-ID: Hs01081199_m1, targeting exon boundary 25/26 of ABCC6), cyclophilin B (PPIB) (Assay-ID: Hs00168719_m1), β-actin (ACTB) (Assay-ID: Hs99999903_m1), GAPDH (Assay-ID: Hs99999905_m1). All RT-PCR experiments were performed at least in duplicate.
Statistic analyses and calculation of gene/pseudogene expression levels
Relative transcript levels were calculated using the ΔΔ-ct method for the two housekeeping genes β-actin and cyclophilin B. Means, p-values and confidence intervals were calculated using the software package SPSS.
This work was supported by a grant from Helse ∅st RHF (200500428-243), Norway, and the Deutsche Forschungsgemeinschaft (KA 1078/4-1), Germany. We thank Runa Marie Grimholt for expert technical assistance.
- Dean M: The genetics of ATP-binding cassette transporters. Methods Enzymol. 2005, 400: 409-429. 10.1016/S0076-6879(05)00024-8.PubMedView ArticleGoogle Scholar
- Kaminski WE, Piehler A, Wenzel JJ: ABC A-subfamily transporters: structure, function and disease. Biochim Biophys Acta. 2006, 1762: 510-524.PubMedView ArticleGoogle Scholar
- Wenzel JJ, Piehler A, Kaminski WE: ABC A-subclass proteins: gatekeepers of cellular phospho- and sphingolipid transport. Front Biosci. 2007, 12: 3177-3193. 10.2741/2305.PubMedView ArticleGoogle Scholar
- Dean M, Annilo T: Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu Rev Genomics Hum Genet. 2005, 6: 123-142. 10.1146/annurev.genom.6.080604.162122.PubMedView ArticleGoogle Scholar
- Mighell AJ, Smith NR, Robinson PA, Markham AF: Vertebrate pseudogenes. FEBS Lett. 2000, 468: 109-114. 10.1016/S0014-5793(00)01199-6.PubMedView ArticleGoogle Scholar
- Balakirev ES, Ayala FJ: Pseudogenes: are they "junk" or functional DNA?. Annu Rev Genet. 2003, 37: 123-151. 10.1146/annurev.genet.37.040103.103949.PubMedView ArticleGoogle Scholar
- Pulkkinen L, Nakano A, Ringpfeil F, Uitto J: Identification of ABCC6 pseudogenes on human chromosome 16p: implications for mutation detection in pseudoxanthoma elasticum. Hum Genet. 2001, 109: 356-365. 10.1007/s004390100582.PubMedView ArticleGoogle Scholar
- Yabuuchi H, Takayanagi S, Yoshinaga K, Taniguchi N, Aburatani H, Ishikawa T: ABCC13, an unusual truncated ABC transporter, is highly expressed in fetal human liver. Biochem Biophys Res Commun. 2002, 299: 410-417. 10.1016/S0006-291X(02)02658-X.PubMedView ArticleGoogle Scholar
- Piehler AP, Wenzel JJ, Olstad OK, Haug KB, Kierulf P, Kaminski WE: The human ortholog of the rodent testis-specific ABC transporter Abca17 is a ubiquitously expressed pseudogene (ABCA17P) and shares a common 5' end with ABCA3. BMC Mol Biol. 2006, 7: 28-10.1186/1471-2199-7-28.PubMedPubMed CentralView ArticleGoogle Scholar
- Vanin EF: Processed pseudogenes: characteristics and evolution. Annu Rev Genet. 1985, 19: 253-272. 10.1146/annurev.ge.19.120185.001345.PubMedView ArticleGoogle Scholar
- Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF, Zeeberg B, Buetow KH, Schaefer CF, Bhat NK, Hopkins RF, Jordan H, Moore T, Max SI, Wang J, Hsieh F, Diatchenko L, Marusina K, Farmer AA, Rubin GM, Hong L, Stapleton M, Soares MB, Bonaldo MF, Casavant TL, Scheetz TE, Brownstein MJ, Usdin TB, Toshiyuki S, Carninci P, Prange C, Raha SS, Loquellano NA, Peters GJ, Abramson RD, Mullahy SJ, Bosak SA, McEwan PJ, McKernan KJ, Malek JA, Gunaratne PH, Richards S, Worley KC, Hale S, Garcia AM, Gay LJ, Hulyk SW, Villalon DK, Muzny DM, Sodergren EJ, Lu X, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madan A, Young AC, Shevchenko Y, Bouffard GG, Blakesley RW, Touchman JW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Krzywinski MI, Skalska U, Smailus DE, Schnerch A, Schein JE, Jones SJ, Marra MA: Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci U S A. 2002, 99: 16899-16903. 10.1073/pnas.242603899.PubMedView ArticleGoogle Scholar
- Jiang Q, Matsuzaki Y, Li K, Uitto J: Transcriptional regulation and characterization of the promoter region of the human ABCC6 gene. J Invest Dermatol. 2006, 126: 325-335. 10.1038/sj.jid.5700065.PubMedView ArticleGoogle Scholar
- Bischof JM, Chiang AP, Scheetz TE, Stone EM, Casavant TL, Sheffield VC, Braun TA: Genome-wide identification of pseudogenes capable of disease-causing gene conversion. Hum Mutat. 2006, 27: 545-552. 10.1002/humu.20335.PubMedView ArticleGoogle Scholar
- Torrents D, Suyama M, Zdobnov E, Bork P: A genome-wide survey of human pseudogenes. Genome Res. 2003, 13: 2559-2567. 10.1101/gr.1455503.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang Z, Harrison PM, Liu Y, Gerstein M: Millions of years of evolution preserved: a comprehensive catalog of the processed pseudogenes in the human genome. Genome Res. 2003, 13: 2541-2558. 10.1101/gr.1429003.PubMedPubMed CentralView ArticleGoogle Scholar
- Consortium IHGS: Finishing the euchromatic sequence of the human genome. Nature. 2004, 431: 931-945. 10.1038/nature03001.View ArticleGoogle Scholar
- Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW: Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics. 2004, 14: 1-18. 10.1097/00008571-200401000-00001.PubMedView ArticleGoogle Scholar
- Zhang Z, Harrison P, Gerstein M: Identification and analysis of over 2000 ribosomal protein pseudogenes in the human genome. Genome Res. 2002, 12: 1466-1482. 10.1101/gr.331902.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang Z, Gerstein M: Identification and characterization of over 100 mitochondrial ribosomal protein pseudogenes in the human genome. Genomics. 2003, 81: 468-480. 10.1016/S0888-7543(03)00004-1.PubMedView ArticleGoogle Scholar
- McCormack WT, Hurley EA, Thompson CB: Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens. Mol Cell Biol. 1993, 13: 821-830.PubMedPubMed CentralView ArticleGoogle Scholar
- Tufarelli C, Hardison R, Miller W, Hughes J, Clark K, Ventress N, Frischauf AM, Higgs DR: Comparative analysis of the alpha-like globin clusters in mouse, rat, and human chromosomes indicates a mechanism underlying breaks in conserved synteny. Genome Res. 2004, 14: 623-630. 10.1101/gr.2143604.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen ZQ, Annilo T, Shulenin S, Dean M: Three ATP-binding cassette transporter genes, Abca14, Abca15, and Abca16, form a cluster on mouse Chromosome 7F3. Mamm Genome. 2004, 15: 335-343. 10.1007/s00335-004-2281-8.PubMedView ArticleGoogle Scholar
- Ban N, Sasaki M, Sakai H, Ueda K, Inagaki N: Cloning of ABCA17, a novel rodent sperm-specific ABC (ATP-binding cassette) transporter that regulates intracellular lipid metabolism. Biochem J. 2005, 389: 577-585. 10.1042/BJ20050159.PubMedPubMed CentralView ArticleGoogle Scholar
- Harrison PM, Zheng D, Zhang Z, Carriero N, Gerstein M: Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability. Nucleic Acids Res. 2005, 33: 2374-2383. 10.1093/nar/gki531.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng D, Zhang Z, Harrison PM, Karro J, Carriero N, Gerstein M: Integrated pseudogene annotation for human chromosome 22: evidence for transcription. J Mol Biol. 2005, 349: 27-45. 10.1016/j.jmb.2005.02.072.PubMedView ArticleGoogle Scholar
- Korneev SA, Park JH, O'Shea M: Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by an antisense RNA transcribed from an NOS pseudogene. J Neurosci. 1999, 19: 7711-7720.PubMedGoogle Scholar
- Duret L, Chureau C, Samain S, Weissenbach J, Avner P: The Xist RNA gene evolved in eutherians by pseudogenization of a protein-coding gene. Science. 2006, 312: 1653-1655. 10.1126/science.1126316.PubMedView ArticleGoogle Scholar
- Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, Soden R, Hayakawa M, Kreiman G, Cooke MP, Walker JR, Hogenesch JB: A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A. 2004, 101: 6062-6067. 10.1073/pnas.0400782101.PubMedPubMed CentralView ArticleGoogle Scholar
- Ross J: mRNA stability in mammalian cells. Microbiol Rev. 1995, 59: 423-450.PubMedPubMed CentralGoogle Scholar
- Zheng D, Gerstein MB: The ambiguous boundary between genes and pseudogenes: the dead rise up, or do they?. Trends Genet. 2007, 23: 219-224. 10.1016/j.tig.2007.03.003.PubMedView ArticleGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la BM, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de JP, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.PubMedView ArticleGoogle Scholar
- Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D: The human genome browser at UCSC. Genome Res. 2002, 12: 996-1006. 10.1101/gr.229102. Article published online before print in May 2002.PubMedPubMed CentralView ArticleGoogle Scholar
- UCSC Genome Browser. http://genome ucsc edu/. 2006, [http://genome.ucsc.edu/]
- Karolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D, Kent WJ: The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 2004, 32: D493-D496. 10.1093/nar/gkh103.PubMedPubMed CentralView ArticleGoogle Scholar
- Rice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000, 16: 276-277. 10.1016/S0168-9525(00)02024-2.PubMedView ArticleGoogle Scholar
- EMBOSS. 2006, [http://emboss.sourceforge.net/]
- TFSEARCH. 2007, [http://www.cbrc.jp/research/db/TFSEARCH.html]
- Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA: Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res. 1998, 26: 362-367. 10.1093/nar/26.1.362.PubMedPubMed CentralView ArticleGoogle Scholar
- Piehler A, Kaminski WE, Wenzel JJ, Langmann T, Schmitz G: Molecular structure of a novel cholesterol-responsive A subclass ABC transporter, ABCA9. Biochem Biophys Res Commun. 2002, 295: 408-416. 10.1016/S0006-291X(02)00659-9.PubMedView ArticleGoogle Scholar
- siRNA at the Whitehead Institute for Biomedical Research. 2004, [http://jura.wi.mit.edu/bioc/siRNAext/]
- Böhringer S, Gödde R, Böhringer D, Schulte T, Epplen JT: A software package for drawing ideograms automatically. Online Journal of Bioinformatics. 2002, 1: 51-59.Google Scholar
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