- Research article
- Open Access
Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression
© Stojic et al; licensee BioMed Central Ltd. 2007
- Received: 06 November 2006
- Accepted: 23 May 2007
- Published: 23 May 2007
The ABCC5 gene encodes an organic anion pump of the ATP-binding cassette (ABC) transporter family, subclass C. The exact physiological function of ABCC5 however is not known. Here, we have isolated three novel ABCC5 splice variants and characterized their role in the regulation of ABCC5 gene expression.
Two additional exons within intron 5 of the ABCC5 gene were identified; one of the exons exhibits alternative donor splice sites. Differential usage of these exons generates three short ABCC5 transcripts named ABCC5_SV1, ABCC5_SV2 and ABCC5_SV3. The variants share the first five exons with the ABCC5 gene but differ in their 3' sequences. ABCC5 and its novel isoforms are abundantly expressed in the human retina. Splice variant ABCC5_SV1 and ABCC5_SV2 contain premature stop codons. While inhibition of nonsense-mediated mRNA decay selectively stabilized ABCC5_SV1 but not ABCC5_SV2, the amount of full length ABCC5 mRNA was simultaneously reduced. A negative regulatory effect on full length ABCC5 expression was also observed when the ABCC5 isoforms were silenced with siRNA duplexes. Finally, we show that the evolutionarily conserved ABCC5_SV2 transcript is translated into a protein abundantly present in endothelial cells of inner retinal blood vessels and along RPE membranes.
Our data suggest that alternative splicing of the ABCC5 gene has functional consequences by modulating ABCC5 gene expression. In addition, at least one ABCC5 splice variant is protein-coding and produces a truncated ABCC5 protein isoform with thus far unknown functional properties in the retina.
- Retinal Pigment Epithelium
- ABCC5 Gene
- Related Multidrug Resistance Protein
- Pseudoxanthoma Elasticum
ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the ATP-dependent translocation of a wide variety of compounds across extra- and intracellular membranes . The substrate diversity ranges from small inorganic ions, amino acids, peptides, sugars, lipids, and anticancer drugs to large proteins. ABC transporters are characterized by a basic modular architecture consisting of two membrane spanning segments and two intracellular nucleotide binding domains with Walker motifs A and B and an ATP-binding cassette signature .
Based on protein sequence homology and phylogenetic analyses, the 56 mammalian ABC transporters have been classified into seven subfamilies with the closely related multidrug resistance proteins (MRPs) grouped together in the C branch of ABC proteins (ABCC). ABCC5 (MRP5) is a typical organic anion pump and belongs to the short type of ABCC proteins which differ from the long type by the lack of an N-terminal transmembrane domain . In vitro transport studies identified ABCC5 as a cellular export pump for numerous compounds including cGMP , nucleoside monophosphate analogs [e. g. [4, 5]], heavy metal compounds and fluorochromes . ABCC5-transfected cells were also reported to exhibit resistance to anticancer and antiviral drugs [5, 7]. The affinity of ABCC5 to its substrates, however, has generally been low. This suggests that the biological significance of ABCC5 as a mediator of active cGMP efflux, its possible role in drug resistance and ultimately its physiological function is still unknown.
Previous mRNA expression studies showed that the ABCC5 gene is widely transcribed among human tissues with highest levels in heart, brain, skeletal muscle, kidney and testis [6, 8]. Multiple mRNA species for various ABCC family members have been described [9, 10] including the ABCC5 locus . Sequencing of a single cDNA clone from a human lung cancer cell line identified a splice variant of ABCC5 formed by the alternative usage of a cryptic donor splice site upstream of exon 11 . This so-called short type of multidrug resistance protein homologue (SMRP) translates into an N-terminally truncated version of ABCC5 (946 versus 1437 amino acids) and shows a similar expression pattern as the full length ABCC5 transcript . The physiological relevance of the rare SMRP transcript is not known.
In this study we have characterized three novel isoforms of the ABCC5 gene generated by alternative splicing of newly identified exons within intron 5 of the ABCC5 gene. The various ABCC5 transcripts are abundantly expressed in the human retina but are also present in many other tissues at varying levels. We provide evidence that alternative splicing of the ABCC5 mRNA may provide an elegant mechanism to achieve a tissue-dependant regulation of ABCC5 gene expression.
Cloning of three novel ABCC5 isoforms
Nucleotide homology searches using the novel ABCC5 exons as templates showed that the sequence and the flanking splice junctions of exon 5A are highly conserved between mammalian species (e.g. 100% sequence identity to monkey/cow and 98% identity to mouse/dog). Analysis of EST sequences from different species confirmed that exon 5A is commonly spliced to exon 5 whereas each species uses different donor splice sites to join exon 5A to downstream sequences within intron 5 of the ABCC5 gene. Thus, sequences overlapping but not identical to human exon 5B are frequently included in alternative transcripts. The presence of ABCC5 splice variants with additional exons within intron 5 of the ABCC5 gene appears to be a common feature among mammalian species possibly indicating a conserved function of these molecules.
Nonsense mediated decay of ABCC5 isoforms
Silencing of the novel ABCC5 isoforms by RNA interference
Localization of ABCC5_SV2 in the retina
Here we characterize three short ABCC5 splice variants which consist of sequences corresponding to the first 5 exons of the ABCC5 gene but revealing distinct 3' ends. The isoforms are generated by the inclusion of one or two novel exons within intron 5 of the ABCC5 gene and the alternative usage of donor splice sites in one of these exons. In-frame translation of the additional exons introduces stop codons, thus generating unique C-termini. Quantitative real time RT-PCR analysis demonstrates that both, the full length ABCC5 transcript and the shorter ABCC5 splice variants are present at varying levels in a number of tissues while all are predominantly expressed in the human retina. Although ABCC5 mRNA expression has repeatedly been found in neurons of the CNS [6, 8], this is the first report of ABCC5 being expressed in the neurosensory retina. ABCC5 can therefore be added to the list of abundant ABC transporters with a function in the eye which for example includes ABCA4, the gene underlying Stargardt's disease  and ABCC6, the gene implicated in pseudoxanthoma elasticum [18, 19]. So far, a role for ABCC5 in retinal disease has not been determined.
Genome-wide analyses have led to the suggestion that alternative splicing affects the vast majority of genes in many organisms [20, 21]. EST-based studies indicated a particularly high level of alternative splicing in neuronal tissues including the retina . The high fraction of splice variants among retinal cDNAs are reflected in numerous reports of alternatively spliced retinal genes [e. g. [13, 23]]. Moreover, retina-specific mRNA processing has been reported for genes with a broader tissue distribution [24, 25]. The retina is a multilayered tissue composed of a number of distinct cell types that are specialized in their function to transform light energy into electric signals. Alternative splicing is regarded an important mechanism to create protein diversity but also to regulate gene expression . Both processes may well be required to perform and control the complex phototransduction process in the retina and also to establish and maintain the structure and integrity of this unique and highly evolved tissue.
Our results on the functional role of alternatively spliced products of the ABCC5 gene in the retina demonstrate that one isoform, ABCC5_SV1, is a target for NMD. NMD is a post-transcriptional surveillance mechanism in eukaryotic cells used to eliminate newly synthesized mRNAs containing premature termination codons (PTCs) [15, 27]. NMD targets which may be generated by mutations or errors in mRNA processing are potentially harmful and need to be cleared. In contrast, alternative splicing to induce NMD is a widely used mechanism for gene regulation, also known as regulated unproductive splicing and translation (RUST) . Our data obtained from NMD inactivation and confirmed by RNA interference show that the expression level of full length ABCC5 transcript is influenced by the presence of alternatively spliced ABCC5 isoforms, in particular ABCC5_SV1. RUST therefore may play a role in ABCC5 gene regulation.
Alternative splicing of genes encoding ABC transporters has previously been reported [9, 10, 29]. Noticeably, alternative splicing of two evolutionarily conserved PTC-containing exons of the ABCC4 gene produces mRNAs that are degraded by NMD . Regulation of ABCC4 gene expression is thought to be accomplished by facilitating the re-initiation of translation. As a consequence shorter ABCC4 proteins lacking a potentially important amino-terminal linker domain would be generated.
The newly identified ABCC5 splice variants encode putative proteins with isoform-specific C-termini that are predicted to be cytosolic. We have generated an antiserum directed against the conserved ABCC5_SV2 isoform which specifically labels the endothelial cells of blood vessels in the inner mouse retina as well as apical and basolateral surfaces of the RPE. This indicates that in addition to gene regulation, alternative splicing of the ABCC5 gene may be a mechanism to increase protein diversity. A polyclonal antibody directed against the C-terminus of the ABCC5 transporter has been widely used to determine the tissue distribution of ABCC5 in several organs. Among other cell types, this antibody strongly stains capillary endothelial cells in the genitourinary tract , in the heart  and in the brain . In brain, a contribution of ABC transporters including ABCC5 to the blood-brain barrier is discussed . Similarly, the ABCC5_SV2 isoform could play a role in the inner and outer blood-retinal barrier function possibly by controlling ABCC5 transporter activity.
Here we show that alternative splicing plays a role in the regulation of ABCC5 gene expression via NMD-related mechanisms. In addition, we present evidence that at least one of the ABCC5 splice variants encodes a functional protein localized to the endothelial cells of the inner retinal blood supply and along RPE membranes. Further studies are needed to determine the precise function of ABCC5 and its regulatory as well as protein-encoding isoforms in the retina. This may also shed light onto a possible contribution of ABCC5 to retinal disease.
Oligonucleotide primers F1 (5'-AGA AGA GCT GAA TGA AGT TG-3'), R1 (5'-TTC AAT GCC CAA GTC AGT G-3'), R (5'-AGC CAT CTA ACA GGT CAT C-3') and R3 (5'-TCA GTA AGA TGG CGG TGC AGT-3') were used to RT-PCR amplify ABCC5 cDNA fragments from human retina (Fig. 1A). The PCR products were directly sequenced utilizing the ABI PRISM Ready Reaction Sequencing Kit and the ABI 310 automated sequencer (PerkinElmer Life Sciences GmbH).
For virtual Northern blot analysis full-length double stranded cDNA was synthesized from 3 μg of total RNA using the SMART cDNA Library Construction Kit (BD Biosciences Clontech) according to the supplier's instructions. Amplification was performed in 19–22 cycles. The cDNAs were separated electrophoretically and transferred to Hybond N+ membrane. A 362 bp DNA fragment from exon 5B of ABCC5 was obtained by RT-PCR amplification with primer pair F (5'-GAA AGA CCC AGA AGG ATG-3')/R and was [α-32P]-dCTP radiolabeled to be used as a probe for filter hybridization at 58°C. The filter was exposed at -80°C for 3 days.
The human total RNA master panel including RNAs from 21 different human tissues was purchased from BD Biosciences. RNA from post-mortem retina and retinal pigment epithelium (RPE) was isolated using the RNeasy Total RNA System Kit (Qiagen). First-strand cDNA was generated from RNA samples by reverse transcription using Superscript II (Invitrogen) and served as a template for subsequent PCR assays. Real-time quantitative RT-PCR (qRT-PCR) was performed as described previously (Krämer et al., 2004). Primer pairs for qRT-PCR analysis and fragment sizes were as follows: ABCC5_SV1 (5'-CAA GAA GAG CTG AAT GAA GT-3' and 5'-ACA GCA CCA AGC AAG TGG TC-3', 147 bp), ABCC5_SV2 (5'-GGC AAG AAG AGC TGA ATG AAG T-3' and 5'-CAG CCA TCC TGA AAA TTT GGT-3', 151 bp), ABCC5_SV3 (5'-GGC AAG AAG AGC TGA ATG AAG T-3'and 5'-CAG TCT CCA AAG GAA GGT GGT-3', 151 bp). Average normalization factors were calculated based on four human housekeeping genes (B2M, TBP, SDHA and HPRT) which displayed a stable expression in all tissues. These factors were then used to determine the relative normalized expression values of the ABCC5 transcript variants. All samples were analyzed in triplicate.
Inhibition of protein synthesis
The RPE cell line ARPE-19 was cultured in DMEM/Ham's F12 (1:1 mixture) supplemented with 2 mM L-glutamine, 15 mM HEPES, 42 mM NaHCO3 and 10% FCS. The retinoblastoma cell line Y79 was grown in DMEM containing 10% FCS. In both cell lines protein synthesis was inhibited by addition of 100 μg/ml puromycin or 100 μg/ml anisomycin (Invitrogen) for 2 hours. The cells were washed with PBS and transferred to medium without antibiotics for another 4 hours. Total RNA was then extracted as described above.
The siRNA duplexes (HPP grade) were purchased from Qiagen. The siRNA.1 was designed to target sequence 5'-AAT TCA GCG TAG CTA CCT CCA-3' and siRNA.2 to target sequence 5'-AAT CTC TCG CCA AGA GTT CAG-3'. A non-targeting siRNA (5'-AAT TCT CCG AAC GTG TCA CGT-3') was used as a control. Cells were seeded in 6-well plates at a density of 1 × 106 cells/well 24 hours prior to transfection. Transfections were performed with 2 μg siRNA per well using the TransMessenger Transfection Reagent (Qiagen) following the manufacturer's suggestions. Total RNA isolated from the cells at 8, 24 and 48 hours after transfection was subjected to qRT-PCR.
An ABCC5_SV2 antiserum (SV2_304) was generated by immunizing rabbits with a GST-ABCC5_SV2 fusion protein containing the unique 11 C-terminal amino acids. The polyclonal antibodies were affinity-purified using a ()HiTrap NHS-activated sepharose HP column (Amersham Biosciences). Mouse posterior eyecups were immersion-fixed in 4% paraformaldeyde in 0.1 M phosphate buffer (PB [pH 7.4]) for 1 hour and cryoprotected in 0.1 M PB containing 18% sucrose for 4 h. The eyecups were embedded in OCT embedding medium (Tissue-Tek), fast frozen in liquid nitrogen and cryosectioned vertically at 10 μm. Cryosections were blocked with 0.1 M PB containing 0.3% Triton X-100 and 10% goat serum for 30 minutes and labeled for 12 hours with SV2_304 diluted 1:100 in 0.1 M PB, 0.1% Triton X-100 and 2.5% goat serum at room temperature. After washing in 0.1 M PB, the sections were incubated with the secondary antibody goat anti-rabbit IgG conjugated to Alexa 488 (Invitrogen) diluted 1:800 for 1 h. Labeled sections were washed in 0.1 M PB, mounted (Confocal Matrix) and examined under an Axioskop-2 mot plus fluorescence microscope (Zeiss). To evaluate the specificity of the SV2_304 antibody, 100 μl of diluted purified antibody was preadsorbed for 4 hours with 100 μg of GST-ABCC5_SV2 fusion protein immobilized on glutathione sepharose beads. The beads were pelleted at 1000 × g for 3 minutes and the supernatants were used for immunohistochemistry as described above.
This work was supported by grants from the Bundesministerium für Bildung und Forschung (BMBF) (01KW9921/0) and the Deutsche Forschungsgemeinschaft (DFG) (We1259/14-3).
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