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Human Molecular Genetics, 2003, Vol. 12, No. 2 99-109
© 2003 Oxford University Press

Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons

Jatinder Kaur Lamba, Masashi Adachi, Daxi Sun, Jaana Tammur, Erin G. Schuetz, Rando Allikmets and John D. Schuetz*

St Jude Children's Research Hospital, Department of Pharmaceutical Sciences, Memphis, TN, USA and Departments of Ophthalmology and Pathology Columbia University, New York, USA

Received August 30, 2002; Accepted October 31, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Drug transporters are an important part of the defense of cells against cytotoxic agents. One major group of transporters is known as multidrug resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the ATP binding cassette transporter superfamily. One family member, ABCC4 (also known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as 9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing to an insertion, and subsequently determined the ABCC4 gene structure. This analysis revealed that the insertion was attributed to two additional exons that would be predicted to produce premature termination codons (PTC) in ABCC4. The highly similar mouse Abcc4 gene also contained these exons, which were remarkable because their size and sequence identity were much higher than the overall similarity between these genes. Further, a comparison of human, monkey and rodent ABCC4 genes revealed that these same PTC-producing exons were also highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons might produce nonsense mRNA, we further tested the hypothesis that these mRNAs were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition selectively stabilized PTC containing ABCC4 transcripts in human, monkey and rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was critically dependent upon protein synthesis, as removal of the inhibitor dramatically decreased expression, which correlated with the resumption of protein synthesis. These are the first studies to indicate that the highly conserved PTC exons of the ABCC4 gene may dictate its expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ATP-binding cassette (ABC) transporters are polytypic, integral membrane transport proteins. Typically the plasma membrane encoded ABC transporters are composed of two nucleotide binding domains (NBDs) and two membrane spanning domains (MSDs). The NBD are highly conserved, whereas the membrane spanning domains MSDs are much more divergent (1). The ABCC subfamily has 12 members with a diverse spectrum of functions (2). For instance, the cystic fibrosis transmembrane conductance regulator (trivial name: CFTR, ABCC7) is a chloride ion channel and mutations in CFTR cause cystic fibrosis (3). ABCC8 and ABCC9 proteins bind sulfonylurea and regulate potassium channels involved in modulating insulin levels (4). Other transporters, such as ABCC1-C3 transport drug conjugates and other organic anions. ABCC4, ABCC5, ABCC11 and ABCC12 are smaller than other ABCC genes and lack an amino terminal MSD domain (2). ABCC4, also known as MRP4, was initially functionally defined by its overexpression and ability to confer resistance to nucleoside analogs including, 9-(2-phoshoenylmethoxyethyl) adenine (5) and 9-(2-phoshoenylmethoxyethyl) guanine, an antineoplastic drug (5). Recently, ABCC4 has been shown to transport ganciclovir nucleotides, which can impact gene therapy with HSV-TK (6). It has also been shown to confer resistance and transports thiopurines, which are effective anti-neoplastics (7). ABCC4 also transports cyclic nucleotides and this agrees with the concept that cyclic nucleotide efflux is part of a biological regulatory loop that occurs when intracellular cAMP levels increase (8). The expression analyses of ABCC4 by northern blot analysis has determined that it is widely expressed and recent studies demonstrate higher expression in the kidney, an important site for cyclic nucleotide excretion and re-absorption (9).

A number of reports have demonstrated alternate splicing of ABCC family members. The earliest report is for MRP1 (ABCC1) (10). The ABCC1 gene contains 31 exons and several alternate spliced forms, which, interestingly enough, retain their reading frames (10). Other ABCC family members express alternatively spliced forms. For instance, the recently described ABCC11 and 12 (aka MRP8 and 9) also contain alternate forms, some of which retain their reading frame, but have lost certain functional domains (11,12). In addition, some contain premature termination codons (PTC), and these variants appear to have lost certain functional domains. It is unknown if these alternate forms impact normal function or represent alternate functional forms.

The gene for human ABCC4 has been localized to chromosome 13q32, but its gene structure is unknown (5,13). Our initial analysis revealed that the ABCC4 gene spanned over 300 kb and had 31 exons (see Fig. 1). However, an unusual ABCC4 cDNA isolated from a lung cDNA library prompted us to further investigate this cDNA and closely analyze the ABCC4 gene structure, which resulted in the discovery of the presence of two additional exons (referred to as 1a and 1b) within the very large (>50 kb) intron 1. These exons disrupt the ABCC4 reading frame because of downstream PTCs. For the purpose of comparative genomics, we isolated the mouse Abcc4 gene which was located on mouse chromosome 14 to a region syntenic to that region on human chromosome 13 containing ABCC4. The mouse Abcc4 gene also had the same overall size and exon organization as the human gene, including these same PTC exons. These PTC exons, their splice junctions and flanking intronic sequences were remarkably conserved among different species. These findings indicate that the primary ABCC4 transcript would contain exons with PTC.



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  		Figure 1. (A) Structural and sequence comparison of the human and mouse ABCC4. Both ABCC4s have 33 exons, and inclusion of exons 1a and/or 1b would not encode functional ABCC4. (B) Percentage nucleotide homology between human and mouse ABCC4 genes. Exons encoding the divergent membrane spanning domains (MSD) and highly conserved Walker motifs and nucleotide binding domains (NBD) have been underlined.

 
The degradative fate of mRNA transcripts containing PTC exons is widely debated as to whether this process initiates in either the cytoplasm (post splicing and nuclear export) or in the nucleus prior to splicing (15–17). Nevertheless, expression of PTC-containing transcripts is often but not always reduced by the process of nonsense-mediated mRNA decay (NMD) (14). In the present study we wanted to explore whether these PTC-containing splice variants of ABCC4 are targets of NMD and further evaluate if this occurs among other ABCC family member splice variants that lack PTC (e.g. ABCC11) or if it confined only to those ABCC family members containing PTC (e.g. ABCC12) (11). The findings reported have strong implications for the expression of ABCC4 in particular.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the human and murine Abcc4 genes
The human ABCC4 was mapped on a radiation hybrid panel between the markers WI-9265 and WI-6695 to chromosome 13q32. Our initial analysis of the genomic DNA of human ABCC4 revealed 31 exons (Fig. 1). Exon sizes ranged from 38 to 250 bp (Table 1). All exons were flanked by GT and AG dinucleotides consistent with the consensus sequences for splice junctions in eukaryotic genes. Of the 30 introns, 17 are class 0, 5 are class 1, and 8 are class 2. The newly identified exons 1a and 1b were class 2 and 1, respectively. The human ABCC4 gene, as the other ABCC subfamily members, contains two membrane-spanning domains (MSD1, MSD2) and two nucleotide-binding domains (NBD1, NBD2). The 374 amino acids constituting MSD1 are encoded by the first eight exons of ABCC4. Each of the NBDs is encoded by four exons, 10–13 for NBD1 and 25–28 for NBD2. The MSD2 is encoded by exons 17–24. Notably, we isolated several ABCC4 cDNAs that, predictions revealed, would not encode a functional protein, due to a 266 bp insertion at the end of exon 1. An interrogation of the NCBI human genome database with this 266 bp fragment revealed that these sequences were surprisingly encompassed within intron 1 (Fig. 1A). Because these exons were found in an mRNA, flanked by GT and AG in the gene and had very good consensus splice donor and acceptor sequences (Fig. 2B), we designated these insertions as exon 1a and exon 1b. The scores for the donor splice site were 0.78 and 0.97 and scores for the acceptor splice site were 0.91 and 0.93 for exons 1a and 1b, respectively (with the ‘splice view’, splice site prediction program). Exon 1a was 113 bp in length and started at position 32008 in the ABCC4 gene [GenBank no. gene (gi 16163337) from NT_029432]. Exon 1b spanned 153 bp in length and started at position 35166 in the ABCC4 gene, a position ~3 kb downstream of exon 1a and 18 kb upstream of exon 2 (Fig. 1A). Mutations in introns of genes can create spurious exons (e.g. CYP3A5) (18). Therefore, it is possible that exon 1a and exon 1b could have been created by mutations within the large ABCC4 intron 1. To assess this possibility, we obtained genomic DNA from liver samples of 26 individuals (see Materials and Methods) and amplified the DNA using primer pairs derived from the flanking intronic regions of exons 1a and 1b that were up to 300 bp away (see Table 2 and Materials and Methods). This distance was selected because it would encompass any variations in splice sites or known sequences affecting splicing (i.e. branch points or exonic/intronic splicing enhancer sequences) (19). Sequencing of these amplified products revealed no interindividual variation in the splice sites, branch points or exons (data not shown) that could explain the insertions. This analysis of over 52 chromosomes from 26 individuals strongly indicates that genetic variation is an unlikely explanation for the presence of exon 1a and 1b.


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  		Table 1. Exon–intron organization of the human ABCC4 gene
 


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  		Figure 2. ABCC4 mRNA containing PTC are found in human, primate and rodent cells. (A) The predicted splice variants produced by different combinations of exon 1a and 1b; the asterisks indicate the presence of PTCs. (B) Alignment of genomic DNA sequences from human, monkey and mouse demonstrated that exons 1a and 1b are highly conserved. The bold and capital letters indicate exon 1a and 1b, the intronic sequences are in small case. The conserved splice junctions are shown in italics. Differences between the sequences are underlined and the stop codons are indicated by the asterisks. (C) Cell lines from different species (human, rat and mouse) were used to look for splice variants of ABCC4. RT–PCR was carried out using total RNA from these cell lines and primers mentioned in the Materials and Methods.

 

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  		Table 2. ABCC4 primers used for amplification of cDNA and genomic DNA
 
Conservation of ABCC4 gene structure
To determine if exon 1a and 1b were phylogenetically conserved, we isolated the mouse Abcc4 gene by screening a BAC library and used the mouse Celera database to verify gene structure (Fig. 1B). We found that the mouse gene is located at chromosome 14 in a region syntenic to human chromosome 13, where the location of human ABCC4 gene resides (5,14). The region encompassing exons 1a and 1b was not available in the mouse Celera database and was generated by directly sequencing this orthologous region in the Abcc4 BAC (nos 27089, 270898 from strain, ES129/SVJ). We then analyzed the murine Abcc4 gene and assessed its splice junctions (Table 3). Overall the mouse gene spanned >350 kb and had the same predicted number and size exons as the human ABCC4 (Table 1). Of the 30 introns, 17 are class 0, 5 are class 1 and 8 are class 2. The newly identified exons 1a and 1b were almost identical to the human gene. These exons were found in murine genomic DNA from other mouse strains (e.g. ICRC Swiss mice and FVB). Finally, a comparison of the length of the introns between mouse and human ABCC4 reveals that of the 30 introns, 25 were almost identical size (see Table 4) and several were almost as large as intron 1 (e.g. intron 19).


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  		Table 3. Exon–intron organization of the murine Abcc4 gene
 

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  		Table 4. Human and mouse Abcc4 intron length
 
Comparison of the nucleotide sequence of exons 1a and 1b revealed that these exons were highly conserved between mouse and human. The percentage nucleotide identity was very high (98–100%) within exons 1a and 1b as compared with the much lower homology for the rest of the coding exons (67–92%) (Fig. 1B). Exon 1a and 1b sequences are more highly conserved than the rest of the ABCC4 gene; however, the possible splice variants (SV1-3, GenBank accession numbers, AY133679, AY133678, AY133680 respectively for SV1, 2 and 3) resulting from the different combinations of exons 1a and 1b reveal that all these insertions disrupt ABCC4's reading frame and produce premature termination of the protein at codon 25 in SV1 and SV3 and at codons 39, 44 and 67 in SV2 (Fig. 2A).

To determine if the ABCC4 exons 1a and 1b were conserved in evolution, genomic DNA from monkey was amplified and sequenced using the primers described (Materials and Methods and Table 2). Figure 2B shows the alignment of the genomic DNA sequences from human (GenBank accession numbers, exon 1a, AF530634, and exon 1b, AF530635), monkey and mouse. The splice junctions of exons 1a and 1b were highly conserved and predicted by each of the splice site prediction programs tested using the default parameters. (www.fruitfly.org/seq_tools/splice.html, http//genio.informatik.uni-stuttgart.de/GENIO/splice/splice.cgi and http//125.itba.mi.cnr.it/~webgene/www.spliceview_ex.html). Exon 1a was 100% identical in human and mouse with a couple of minor differences in the monkey sequence (underlined in the Fig. 2B). Human and monkey sequences for exon 1a were 91% identical (Fig. 2B). The exon 1b sequence from monkey shared complete sequence identity with the human sequence while for the mouse sequence there was 98% identity with the human. Thus, among different species of mammals, the ABCC4 gene shares a high degree of sequence identity within exon 1a and 1b, the flanking introns and splice junctions. The high conservation of exons 1a and 1b from mouse to man indicates that this ‘exonic’ inactivation (the inclusion of exons with PTC) of the ABCC4 gene occurred before these species diverged and indicates a common ancestor and perhaps function.

Exon 1a and 1b are expressed
Although we identified a ABCC4 cDNA with PTCs, it remained possible that exon 1a and exon 1b are either not or rarely expressed in vivo. Therefore, using conserved ABCC4 primers (see Materials and Methods) we amplified the region between exon 1 and exon 2 in multiple species (Fig. 2C). These primers would detect spliced ABCC4 products of 194, 460, 347 and 307 bp, which represent WT (predicted to encode a functional protein), SV1, SV2 and SV3 gene products, respectively. These splice variants would produce downstream termination codons in the following positions for the human (codon 25 in SV1 and 3, codons 39, 44, 67 in SV2) and for the monkey (same codons as humans due to the high degree of homology with monkey) and the mouse. Human (Saos-2), primate (COS7, included in Fig. 3B) and rodent (rat, H35 and mouse, NIH3T3) RNA was evaluated for the presence of the ABCC4 splice variants. All cell lines from each species had multiple splice variants, although some splice variants were predominant (e.g. SV2>SV1>SV3, see Fig. 2C). We used direct sequencing to confirm the nucleotide identity of SV1-3 from K562 cDNAs (see Fig. 3).



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  		Figure 3. Protein synthesis inhibition selectively increases PTC containing ABCC4 RNA. (A) Four human cell lines; (B) one monkey and (C) one rodent cell line was treated with 100 µg/ml Puromycin (P, all cell lines) and 100 µg/ml anisomycin (A, human cell lines) for 4 h. Marker lane (M) represents 100 bp DNA ladder. RNA was isolated and the RT–PCR was performed. Protein synthesis was inhibited by more than 90% after treatment with the drugs (not shown). These are representative gels from experiments repeated multiple times with similar results (n=2–4).

 
We had observed that the size of many of the introns is conserved between mouse and human ABCC4. The largest conserved introns were 19, 20, 4 and 8, and they ranged in size from the largest 45 kb human (41 kb mouse) to the smallest, i.e. intron 8, 11 kb in human and 8.5 kb in mouse. To test the possibility that ABCC4 splice variants occur in large introns we amplified RNA using ABCC4 primers that amplified a region between exon 13 and 23. We found a single PCR product of 1 kb and did not detect insertion of unidentified exons into these PCR products. This indicates that a long ABCC4 intron, alone does not produce splice variants.

ABCC4 splice variants undergo nonsense mediated mRNA decay
The three ABCC4 splice variants SV1, SV2 and SV3 have PTC (Fig. 2). Normally, mRNAs that have PTC are low in abundance and degradation of these mRNAs occurs through a surveillance mechanism referred to as NMD, which can be inactivated by blocking protein synthesis (16). However, not all PTC mRNAs are targeted for decay and degradation may occur via alternate mechanisms (14). We determined if ABCC4 PTC-containing transcripts were subject to NMD and if this process was conserved for ABCC4. The protein synthesis inhibitors puromycin and anisomycin were used in the following studies with cycloheximide excluded for reasons described below.

Puromycin and anisomycin selectively stabilize the ABCC4 nonsense transcripts, without causing a dramatic effect on the wild-type ABCC4 transcripts, whereas cycloheximide stabilizes all ABCC4 transcripts (WT and SVs), most likely by binding to and stabilizing the ribosomal mRNA complex (20). Therefore we used puromycin and anisomycin. Human cell lines were treated with either puromycin or anisomycin whereas, for convenience, monkey and rodent cell lines were treated with puromycin. Total cellular RNA was isolated and RT–PCR conducted to assess the level of the ABCC4 splice variants. Treatment of human cell lines with these protein synthesis inhibitors increased the intensity of multiple splice variants (SVs) of ABCC4 in all the human cell lines evaluated (H1299, K562, CACO2 and Saos-2) (Fig. 3A). Some of the splice variants strongly increased relative to others, which may be due to tissue-specific splicing effects (21,22). Throughout the study we observed that SV2 was the predominant splice variant as compared to SV1 and 3. To determine the fold increase in SV2 by puromycin we performed real-time PCR analysis and discovered that puromycin treatment increased SV2 greater than 60-fold, thus demonstrating that SV2 mRNAs increase to substantial levels in the absence of protein synthesis. The selective induction of the nonsense encoding ABCC4 splice variant was also observed in the monkey and rodent cell lines (Fig. 3B and C) after treatment with puromycin. These studies, demonstrating evolutionary conservation of ABCC4 gene structure and induction of PTC containing ABCC4 by protein synthesis inhibition, indicate that multiple alternately spliced ABCC4 PTC-containing transcripts are regulated by NMD.

Some ABCC genes have alternatively spliced forms that lack PTCs (e.g. ABCC11) while others contain termination codons upstream of exon–exon junctions (e.g. ABCC12), therefore we tested if inhibition of protein synthesis affected the level of an ABCC11 alternate spliced form (11). We designed ABCC11 primers to concurrently amplify the wild-type and the SV from RNA isolated from MCF7, CACO2 and the H1299 cell lines that had been treated with puromycin as described above (see Fig. 3). As expected we found that puromycin treatment had no effect on the levels of the ABCC11 SV in MCF7 (Fig. 4A). Similar results were obtained for CACO2 and H1299 cell lines (not shown). These findings indicate that the steady state level of the ABCC11 SV is not regulated by protein synthesis inhibition. In contrast, ABCC12 has several splice variants (A–D) (11), each of which contains a PTC. We evaluated whether the levels of these splice variants were controlled by protein synthesis and found that inhibition of protein synthesis led to increased A, B and D expression (C was not evaluated), but have only shown splice variant B (Fig. 4B) because it appears to be the most abundant. Cumulatively, these studies indicate that the presence of a PTC codon in an exon is essential for increasing the level of expression of ABCC12 after protein synthesis inhibition.



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  		Figure 4. Protein synthesis inhibition does not increase ABCC11 splice variant, but does increase an ABCC12 splice variant containing a premature termination codon. MCF7 cell line was treated with 100 µg/ml Puromycin (P) for 4 h. RNA was isolated and the RT–PCR was preformed. Protein synthesis was inhibited by more than 90% after treatment with the drugs (not shown). (A) ABCC11 splice variant A is shown as SV and is due to a deletion in exon 28; (B) ABCC12 splice variant B is shown, although it should be noted that the other splice variants tested (A and D ) were also increased by protein synthesis inhibition (not shown).

 
Translation arrest stabilizes mRNA containing PTCs, while the re-initiation of protein synthesis abrogates the stabilization of these mRNAs. To determine whether the PTC ABCC4 mRNA was specifically regulated by protein synthesis, H1299 and CACO2 cell lines were either untreated or treated with puromycin for 2 h or treated with puromycin for 2 h followed by removal of puromycin and further incubation in drug-free medium for an additional 4 h. Subsequently, the amounts of ABCC4 WT and SVs were determined by RT–PCR and protein synthesis was determined in parallel cultures using a pulse-labeling technique (Fig. 5) to measure the amount of 3H-Leu incorporation. For both the CACO2 and H1299 the amount of splice variants increases almost two-fold after treatment with puromycin (lane 2 compared with control, lane 1). However, 4 h after removal of puromycin (lane 3), the level of SV has returned to pre-treatment levels. This is consistent with the rapid turnover of nonsense mRNAs and indicates that the half-life of ABCC4 transcripts containing PTC is strongly controlled by translation.



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  		Figure 5. The abundance of PTC-ABCC4 is inversely related to protein synthesis. Human cell lines (CACO-2 and H1299) were treated for 2 h with 100 µg/ml puromycin (lane labeled ‘P’), the medium was changed after 2 h of puromycin treatment and the cells were grown in the medium without puromycin for 4 h (lane labeled ‘postP’) and the inhibition of protein synthesis was monitored by assaying incorporation of 3H leucine, RNA was isolated from cells that were untreated (lane labeled ‘-’), treated with puromycin (lane labeled ‘P’) or after its removal (lane labeled ‘post P’). Splice variants of MRP4 were analyzed by RT–PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study we have determined that the human and mouse ABCC4 gene have the same overall structural organization and that this has strong implications for its expression. This in combination with the fortuitous detection of an unusual human ABCC4 cDNA led us to identify two additional exons within the ABCC4 gene. These exons were present in intron 1 and designated as exon 1a and exon 1b. Both exons had consensus splice sites, and were more highly conserved than the rest of the exons in murine and human ABCC4. The presence of either one or both of these exons resulted in three major splice variants. Notably, the inclusion of exon 1a and/or exon 1b in any combination produced a frame shift and premature termination codon. No genetic variation in ABCC4 gene could account for the presence of these exons. In addition, no simple property of ABCC4 such as a long intron appeared to correlate with the production of additional ABCC4 nonsense encoding transcripts. We also demonstrated that these newly discovered splice variants of ABCC4 with premature termination codons are targets for NMD. Although the molecular mechanism of NMD remains controversial, it has been observed that interference with the efficiency of translation can block NMD (23–26). Our experiments with human, monkey and rodent cell lines revealed that the PTC–ABCC4 transcripts increased after treatment with protein synthesis inhibitors. Moreover, we showed that the amount of the PTC–ABCC4 transcripts was absolutely dependent upon protein synthesis, thus revealing a classic characteristic of NMD (27). The evolutionary conservation of these ABCC4 PTC exons suggests at least two possibilities: (1) the protein encoded either 5' or 3' of these exons plays an important functional role; or (2) the nucleotide sequences in or around these exons contain cis-elements that are critical to selecting exons in the ABCC4 gene.

It is likely that conserved cis-elements are critical for selecting the PTC exons in processed ABCC4. For instance, throughout our studies we observed that SV2 was predominant compared with SV1 and 3. The exact reason for this is currently unknown. However, a likely explanation is that both SV1 and SV3 contain exon 1a and it contains a much weaker splice site than exon 1b (see Results). Thus, we speculate that transcripts containing exon 1a are generated less frequently during RNA splicing. In contrast, exon 1b contains better splice sites and would be predicted to become a predominant transcript.

We found no interindividual variation associated with the inclusion of the ABCC4 PTC exons and propose that the ABCC4 exons 1a and 1b play a role in regulating its expression. This could occur by the ABCC4 PTC exons facilitating translation re-initiation and is likely since the levels of nonsense mRNAs are at levels up to 30% of the normal message (28,29). One example of translation re-initation is found in the mdm2 gene in which a novel exon (mdm2 alpha) containing a PTC facilitates translation re-initiation (30). Moreover, the production of the mdm2 alpha transcript is regulated because it occurs only in select tissues and more importantly has a biological function in that it is unable to interact with p53 (30), a role that may contribute to mdm2 being oncogenic. The possibility that translation re-initiation in ABCC4 transcripts containing PTCs occurs is unknown at this point. Nevertheless, the cis properties of the ABCC4 transcripts containing PTC-exons are favorable for translation re-initiation. We note that in exon 2 of ABCC4 the methionine at codon 44 (Met 44) is conserved between mouse and man and that it has properties required for translation re-initiation such as a conserved upstream open-reading frame and the lack of a consensus Kozak sequence (A/GxxATGG). Finally, because translation can abrogate NMD (31), it is possible that the cellular extent of translation re-initiation may determine the level of alternate forms of ABCC4.

The possibility that protein sequences upstream or downstream of ABCC4 are intimately linked to a functional aspect of ABCC4 containing PTC exons seems likely if we compare the known functional domains in the proteins encoded by the ABCC family. Specifically, ABCC4, like other ABCC family members, contains an amino-terminal linker domain, Lo (2). In ABCC1, this domain is essential for transport function and may facilitate membrane association (32). It is notable that the ABCC4 Lo contains a highly structured amphipathic helix of 13 amino acids spanning residues 24–36 and that this is highly conserved among ABCC1-6 (32). If ABCC4 re-initiates translation at Met 44 then this domain would be removed, but the truncated ABCC4 would be almost full length. The removal of the Lo domain in ABCC1 creates a protein that is incapable of transport and localizes cytoplasmically (32). Currently, it is unknown if this domain has any significant role in ABCC4 function or localization. We suspect that it might because ABCC4 is relatively unique in that it lacks the terminal membrane spanning domain, (MSDo) found in ABCC1-3, 6 (2).

Genes have been found to undergo tissue specific alternate splicing, e.g. alternate splicing of exon 1 of neuronal nitric oxide synthase (nNOS) gene controls tissue and developmental stage-specific expression (21); alternate splicing of CYP4F3 with inclusion of either exon 3 or exon 4 results in enhanced protein diversity to accommodate different substrates as well as impact tissue specific expression (33). If the conserved PTC exons facilitate translational re-initiation then the alternate forms of ABCC4 might contribute to protein diversity and influence the type of ABCC4 expressed. We propose that the cellular environment regulates ABCC4 splicing and provides a checkpoint, after transcription, for fine tuning and selecting the type of ABCC4 required. Although it is unknown at this point, such a checkpoint may serve to allow a cell to determine which form of ABCC4 is functionally required and then modulate its expression accordingly.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The human lung cDNA library was purchased from Clontech (Palo Alto, CA, USA) ({lambda}triplEx, catalog no. HL5030t, pooled samples from two Caucasian females).

Human ABCC4 gene
The exon/intron structure of human ABCC4 gene was determined by direct sequencing of bacterial artificial chromosome (BAC) clone(s) AL356257, AL157818, AL139381, by PCR amplification of genomic DNA, and by computer searches of the nr, htgs, gss and dbEST databases with the BLAST program on the NCBI file server. The structure of the 5' end of the gene was determined with the GenomeWalkerTM kit (Clontech) according to the manufacturer's protocol.

Primers for amplification of genomic fragments were designed from the ABCC4 cDNA sequence (AYO81219 and XM_036453). The positions of introns were determined by comparison between genomic and cDNA sequences.

GenBank sequence comparisons
The human ABCC4 gene sequence was obtained from the working draft sequence of chromosome 13 (GenBank no. NT_029432). ABCC4 gene was localized in the region spanning 203207-515370 (GenBank no. gi 1616337, gene in NT_029432).

The ABCC4 5'-flanking region (nucleotides -1 to -1435 with respect to translation start site) was analyzed with the program NNPP/eukaryotic to predict the transcription-initiation site. The nearest transcription initiation site to ATG codon was determined as position -158 (score 0.90). Putative binding sites for transcription factors were identified with TRANSFAC program (34). Several potential binding sites for transcription regulatory factors such as Ap1, Ap2, Ap4, C/EBP, NF-kB and Sp1 were identified. The consensus sequences were not detected for the TATA-box. It is likely that the human ABCC4 (MRP4) gene, similarly to human ABCC1 (MRP1), rat ABCC2 (Mrp2), human ABCC2 (MRP2) and ABCC3 (MRP3), is under the control of a TATA-less promoter (35).

Mouse Abcc4 gene
Subsequently, the partial mouse Abcc4 gene sequence was obtained from the Celera database (GA_x5J8B7W5V5A: 1 500 000). The sequence of the entire mouse Abcc4 gene was determined by a combination of long-range PCR using genomic DNA as template, rapid amplification of cDNA ends (RACE), and direct sequencing of BAC clones [nos 27089, 270898 which had been isolated after screening a mouse BAC library (Genome Systems, St Louis, MO, USA) with a partial murine Abcc4 cDNA]. In addition, human ABCC4 primers from conserved gene regions were used to amplify mouse Abcc4 fragments from mouse testis Marathon-Ready cDNA library (Clontech). A database search of the proprietary Celera Discovery SystemTM mouse database and GenBank htgs database with the human ABCC4 cDNA as a probe and the BLAST program on the NCBI file server was utilized to assemble the cDNA and to determine the genomic organization of the mouse Abcc4 gene. The degree of homology between mouse and human gene sequence was assessed by directly comparing the genes exon-by-exon. The sizes of the introns were determined by calculating the distance between adjacent exons using the map positions assigned in the Celera Discovery System mouse database.

Cell lines
We utilized five different human cell lines. The cell lines used were as follows: K562 (erythroleukemia), H1299 (lung carcinoma), MCF7 (breast carcinoma), Saos-2 (osteosarcoma) and CACO2 (colon carcinoma). The monkey and rat cell lines used were COS7 and H35, respectively. The mouse cell lines, HepaIc1c7 and NIH3T3 were also used in the present study. K562, H1299, MCF7 cell lines were grown in RPMI 1640 (BioWhittaker, Walkersville, MD, USA) supplemented with 10% FBS (BioWhittaker), 1% pencillin and streptomycin (10 000 units/ml penicillin and 10 000 µg/ml streptomycin, Invitrogen, Carlsbad, CA, USA), and 1% glutamine (200 mM, Invitrogen). CACO2, Saos-2, Cos7 and NIH3T3 were grown in DMEM (BioWhittaker) and HepaIc1c7 and H35 were grown in MEM (Invitrogen) supplemented as mentioned above. All the cell lines were maintained in a 5% CO2 incubator at 37°C.

Protein synthesis inhibition
The extent of inhibition of protein synthesis was estimated by assaying the radioactivity associated with acid-insoluble fraction after a pulse-label with [3H] Leucine (36). Briefly, 4 µ Ci [3H] leucine were added and cells were incubated for 1 h at 37°C. The cells were then washed with ice-cold phosphate buffered saline and lysed with 5% trichloroacetic acid (TCA). After a 30 min incubation on ice, the lysate was spun to precipitate the acid-insoluble material. The supernatant was discarded and the pellet was washed twice with ice cold 5% TCA and then hydrolyzed using 1.0 N NaOH at 37°C for 1 h. The hydrolysate was measured for radioactivity by scintillation counting and protein was quantified by the method of Lowry et al. (37). In the present study we used puromycin, anisomycin and cycloheximide as protein synthesis inhibitors (Sigma-Aldrich, St Louis, MO, USA). We found that protein synthesis was inhibited by more than 90% after a 2 h incubation with drug at the following concentrations: puromycin, 100 µg/ml; cycloheximide, 10 µg/ml; anisomycin, 100 µg/ml. Treatment for 2–4 h was selected.

ABCC4 splice variants identified by RT–PCR
RNA was isolated according to the manufacturer's protocol using the TRIZOL Reagent (Invitrogen). First-strand cDNA was synthesized using Superscript first-strand synthesis system (Invitrogen) with oligo dT as a primer. Amplification of the ABCC4 cDNA was carried out using primers that annealed to exon 1 and exon 2 (see Table 2). The forward primer MRP4-1F1 anneals to exon 1 and the reverse primer MRP4-2R1 to exon 2. Amplification of the cDNA was conducted as follows: 2 µl of the cDNA, 10 pmol of each primer, 0.2 mM dNTPs, 1xPCR buffer and 1.75 units of Taq DNA polymerase (Expand High Fidelity PCR system, Roche, Indianapolis, IN, USA). The PCR was carried on MJ Research thermal cycler (MJ Research Inc., Watertown, CA, USA) with an initial denaturation step at 92°C for 3 min, followed by 32 cycles of denaturation at 92°C for 30 s, annealing at 60°C for 30 s and synthesis at 72°C for 1 min. To ensure complete PCR products a final synthesis step at 72°C for 5 min was included. These primers would produce the following products: 194 bp fragment containing no insertions, 307 bp fragment if exon 1a was inserted, a 347 bp fragment if only exon 1b was inserted and 460 bp if both exons 1a and 1b were included. Real-time PCR for the semi-quantification of the SV2 splice variant was carried out using the QuantiTectTM SYBR Green® Green PCR kit following the manufacturer's instruction (Qiagen, Charsworth, CA, USA). The primers were designed so that only SV2 would be amplified. Amplification was carried on the ABI PRISM 7700 (PE Applied Biosystems) using primers MRP4-Taq-F1 and MRP4-2R1 (see Table 2). PCR consisted of an initial activation step at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 30 s, 60°C for 30 s, and synthesis at 72°C for 1 min. The amount of the SV2 formed was normalized to GAPDH. Amplification of ABCC11 cDNA from CACO2, MCF7 and H1299 was also performed. The primers used are as follows: ABCC11-F1, 5'-AGGAACCATCAGATTCAACCT-3' and ABCC11-F2, 5'-CTCAGTGAAGAAGTGGCTGT-3'. For ABCC12 splice variants A, B and D were amplified. The primers for ABCC12 splice variant B were: forward primer, ABCC12-2908F 5'-GTACACTGCAAGCATGGTGT-3'; and reverse primer, ABCC12-3360R 5'-TGATGCAGCTCTCCTTCTTGC-3'. For splice variants A, and D the primers used were: forward primer ABCC12-3703F 5'-GATCACCTTCAGAGACTATCA-3'; and reverse primer ABCC12-4199R 5'-TCAGTCTTGGAGTCCATAGAGG-3'. The PCR amplification was carried out at 57°C for 36 cycles using 2 µl of the cDNA from either the untreated and treated cells.

Amplification of the genomic DNA
Genomic DNA from the liver samples of individuals with (n=10) and without the spliced variants (n=16) was isolated using DNeasy Tissue kit (Qiagen) and analyzed for the presence of SNPs within and the flanking regions of exons 1a and 1b.

Exon 1a was amplified by PCR using primers MRP4G-F2, which anneals 310 bp upstream of the exon 1a 5' splice site, and MRP4G-R2, which anneals 305 bp downstream of the exon 1a 3' splice site (Table 2). Genomic DNA (50 ng) was used as a template. PCR was carried out in a reaction mixture containing 10 pmol of each primer, 0.2 mM dNTPs, 1xPCR buffer and 1.75 units of Taq DNA polymerase (Expand High Fidelity PCR system, Roche). The PCR amplification conditions were as follows: an initial 5 min denaturation step at 92°C, followed by 32 cycles composed of 92°C denaturation for 30 s, an annealing step at 55°C for 30 s and a synthesis step at 72°C for 1 min. A final single 72°C extension step was also conducted for 5 min.

Amplification of exon 1b was carried out using primers MRP4G-F4 (Table 2), which annealed to the genomic DNA 154 bp upstream of the exon 1b 5' splice site and MRP4G-R4 , which annealed to genomic DNA 321 bp downstream of exon 1b 3' splice site. The composition of the reaction and the cycling conditions was identical to above with the exception that the annealing step was at 60°C for 30 s.

Amplification of exon 1a from monkey and mice genomic DNA (Promega, Madison, WI, USA) was carried out using the forward primer MRP4G-F3 which was designed from the human ABCC4 gene (GenBank no. gene gi16163337 in sequence NT_029432) and the MRP4G-R2. Exon 1b was amplified using the primers and conditions mentioned above for amplification from human genomic DNA.

To directly sequence the PCR products the unincorporated nucleotides and primers were removed by incubation with Shrimp Alkaline Phosphatase (1 unit, USB, Cleveland, OH, USA) and Exonuclease I (5 unit, USB) for 30 min at 37°C. Following thermal denaturation of the enzymes (80°C for 15 min) sequence analysis was carried out on an ABI Prism 3700 Automated Sequencer using the PCR primers.

Sequence analysis
The nucleotide sequences were assembled using the Phred-Phrap-Consed package (University of Washington, Seattle, http://droog.mbt.washington.edu/PolyPhred.html) specifically developed to detect the presence of heterozygous single nucleotide substitutions by fluorescence based sequencing of PCR products (38,39). The following programs were used for prediction of splice sites: www.fruitfly.org/seq_tools/splice.html, http//genio.informatik.uni-stuttgart.de/GENIO/splice/splice.cgi and http//125.itba.mi.cnr.it/~webgene/wwwspliceview_ex.html


   ACKNOWLEDGEMENT
 
This work was supported by National Institutes of Health research grants GM-60904, GM-60346, GM-61393 and P30 CA-21765, and by the American Lebanese Syrian Associated Charities (ALSAC). We thank Dr William Evans for his critical and insightful comments and Dr Piet Borst for his suggestion to determine whether the ABCC4 gene insertion was evolutionarily conserved.


   FOOTNOTES
 
* To whom correspondence should be addressed. Fax: +1 9015256869; Email: john.schuetz{at}stjude.org Back


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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