BACKGROUND
atp-binding cassette  transporters are integral membrane proteins which mediate the atp-dependent translocation of a wide variety of compounds across extra- and intracellular membranes  <cit> .
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  <cit> .
based on protein sequence homology and phylogenetic analyses, the  <dig> mammalian abc transporters have been classified into seven subfamilies with the closely related multidrug resistance proteins  grouped together in the c branch of abc proteins .
abcc <dig>  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  <cit> .
in vitro transport studies identified abcc <dig> as a cellular export pump for numerous compounds including cgmp  <cit> , nucleoside monophosphate analogs , heavy metal compounds and fluorochromes  <cit> .
abcc5-transfected cells were also reported to exhibit resistance to anticancer and antiviral drugs  <cit> .
the affinity of abcc <dig> to its substrates, however, has generally been low.
this suggests that the biological significance of abcc <dig> 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 abcc <dig> gene is widely transcribed among human tissues with highest levels in heart, brain, skeletal muscle, kidney and testis  <cit> .
multiple mrna species for various abcc family members have been described  <cit>  including the abcc <dig> locus  <cit> .
sequencing of a single cdna clone from a human lung cancer cell line identified a splice variant of abcc <dig> formed by the alternative usage of a cryptic donor splice site upstream of exon  <dig>  <cit> .
this so-called short type of multidrug resistance protein homologue  translates into an n-terminally truncated version of abcc <dig>  and shows a similar expression pattern as the full length abcc <dig> transcript  <cit> .
the physiological relevance of the rare smrp transcript is not known.
in this study we have characterized three novel isoforms of the abcc <dig> gene generated by alternative splicing of newly identified exons within intron  <dig> of the abcc <dig> gene.
the various abcc <dig> 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 abcc <dig> mrna may provide an elegant mechanism to achieve a tissue-dependant regulation of abcc <dig> gene expression.
RESULTS
cloning of three novel abcc <dig> isoforms
large-scale sequencing of a human retinal cdna library  <cit>  revealed two small cdna clones, 6d2f  and a06-ret <dig> , located within intron  <dig> of the abcc <dig> gene .
subsequent blastn database queries  <cit>  revealed the retinal sequences to correspond to the 3' end of three overlapping full length cdna clones isolated from cancer cells and placenta .
after assembly, the human mrna sequences from genbank comprise the first five exons of the abcc <dig> gene spliced to two alternative exons within intron  <dig> of the abcc <dig> gene, named exon 5a and 5b .
to further explore the existence of novel abcc <dig> splice variants in retina, we performed rt-pcr on human retinal rna with primers located in exon  <dig> and exon 5b.
three distinct pcr products of  <dig>   <dig> and  <dig> base pairs  were repeatedly amplified .
direct sequencing of the pcr products showed that the two smaller fragments differ by the inclusion of exon 5a.
in addition, the usage of an alternative splice donor sequence produces a larger exon 5a  giving rise to the  <dig> bp fragment .
rt-pcr with primers located in exon  <dig>  and exon  <dig>  amplified a single  <dig> bp fragment lacking exon 5a, 5aa or 5b  indicating that the newly identified exonic sequences represent the respective 3'-ends of novel abcc <dig> splice variants abcc5_sv <dig>  , abcc5_sv <dig>   and abcc5_sv <dig>   .
this is confirmed by the presence of a potential consensus polyadenylation signal, aataaa, within  <dig> bases of the 3' end of exon 5b.
translation of the putative abcc5_sv <dig>  abcc5_sv <dig> and abcc5_sv <dig> mrna sequences revealed stop codons in exon 5aa, exon 5a and exon 5b, respectively .
this should lead to putative truncated abcc <dig> proteins which share the  <dig> n-terminal amino acids with the full length abcc <dig> transporter and terminate in short distinct c-termini of  <dig> ,  <dig>  and  <dig>  amino acids in length.
nucleotide homology searches using the novel abcc <dig> exons as templates showed that the sequence and the flanking splice junctions of exon 5a are highly conserved between mammalian species .
analysis of est sequences from different species confirmed that exon 5a is commonly spliced to exon  <dig> whereas each species uses different donor splice sites to join exon 5a to downstream sequences within intron  <dig> of the abcc <dig> gene.
thus, sequences overlapping but not identical to human exon 5b are frequently included in alternative transcripts.
the presence of abcc <dig> splice variants with additional exons within intron  <dig> of the abcc <dig> gene appears to be a common feature among mammalian species possibly indicating a conserved function of these molecules.
expression analysis
qualitative rt-pcr analysis revealed varying transcription levels of the novel splice variants in human retina with an increase of expression from abcc5_sv <dig> to abcc5_sv <dig> .
the tissue distribution of the novel abcc <dig> splice variants was studied by virtual northern blot analysis with a radiolabeled probe representing exon 5b.
a single strong band of approximately  <dig>  kb was detected exclusively in retina but not in bladder, heart, lung, skeletal muscle and rpe .
to further investigate the relative expression of the splice variants, quantitative real-time rt-pcr  with primer pairs specific for each of the abcc <dig> transcripts was performed with rna extracted from ten human tissues including samples from three distinct brain areas .
an overall abundant expression of abcc <dig> and the shorter abcc <dig> isoforms was observed in human neural retina.
in addition, full length abcc <dig> is expressed at moderate levels in several tissues including brain, rpe, bladder and heart.
a relatively high expression in rpe, bladder and stomach was found for abcc5_sv <dig> whereas abcc5_sv <dig> and abcc5_sv <dig> are only weakly present in nonneuronal tissues.
moderate expression of abcc5_sv <dig> and abcc5_sv <dig> was detected in cerebellum.
taken together, our data show that each tissue analyzed expresses the various abcc <dig> transcripts but at distinct levels suggesting a complex regulation of gene expression.
nonsense mediated decay of abcc <dig> isoforms
a stop codon which resides over 50– <dig> nucleotides upstream from a downstream exon/exon junction is predicted to be recognized by the translational machinery as a premature termination codon  and thus should be targeted by nonsense mediated mrna decay   <cit> .
splice variant abcc5_sv <dig> contains a stop codon  <dig> bp upstream of the exon 5aa/5b splice junction fulfilling the criteria for a ptc.
to determine whether the abcc5_sv <dig> mrna is subject to nmd we inactivated the nmd surveillance mechanism by the addition of translation inhibitors puromycin, or alternatively anisomycin,  <cit>  to human cell lines arpe- <dig> and y <dig> which were shown before to express abcc <dig>  abcc5_sv <dig>  abcc5_sv <dig> and abcc5_sv <dig> .
by qrt-pcr, puromycin treatment drastically increased the relative expression of abcc5_sv <dig> mrna by almost 3-fold in the arpe- <dig> cell line and by more than 2-fold in the y <dig> cell line .
interestingly, in both cell lines the expression of the abcc <dig> transcript decreased by a factor of approximately two.
in contrast, the relative expression levels of abcc5_sv <dig> and abcc5_sv <dig> remained unaffected by the addition of the translation inhibitors.
the concentration of the mrnas largely returned to pre-treatment levels after protein synthesis was restored by removing puromycin from the media.
similar to the treatment with puromycin, application of anisomycin lead to a 2- and 4-fold increase of abcc5_sv <dig> mrna in y <dig> and arpe- <dig> cells, respectively, whereas the levels of the remaining abcc <dig> transcript variants were stable  or decreased  .
together, these data demonstrate that inhibition of cellular protein synthesis leads to a selective stabilization of the abcc5_sv <dig> mrna indicating that this transcript is regulated by nmd.
in addition, elevated levels of abcc5_sv <dig> mrna appear to regulate abcc <dig> gene expression without exerting any effects on the remaining short abcc <dig> isoforms.
silencing of the novel abcc <dig> isoforms by rna interference
to further investigate whether the expression of the various abcc <dig> transcripts may be co-regulated, abcc5_sv <dig>  abcc5_sv <dig> and abcc5_sv <dig> were silenced by rna interference.
consequently, treatment of arpe- <dig> cells with an sirna probe targeted to exon 5b  decreased the expression of abcc5_sv <dig>  abcc5_sv <dig> and abcc5_sv <dig> up to 80% while the expression of abcc <dig> increased  <dig> -fold over time .
a similar rise in abcc <dig> expression was observed when abcc5_sv <dig> and abcc5_sv <dig> expression was selectively suppressed by sirna.
<dig> .
the level of abcc5_sv <dig> essentially remained unaffected.
sirna probes to specifically target abcc5_sv <dig> without affecting the other two isoforms were designed, but consistently failed to reveal a measurable knock down effect of abcc5_sv <dig> expression .
from our results we conclude that abcc <dig> mrna expression is differentially regulated by the presence of one or more short abcc <dig> splice variants, most likely by abcc5_sv <dig> and/or abcc5_sv <dig>
localization of abcc5_sv <dig> in the retina
the most c-terminal  <dig> amino acid peptide specifically encoded by the abcc5_sv <dig> isoform appears highly conserved in a number of mammalian species suggesting a functional constraint on this sequence .
to investigate whether abcc5_sv <dig> indeed is expressed as a short abcc <dig> protein isoform, we generated polyclonal antibodies  directed against its unique  <dig> amino acid terminus.
immunofluorescence labeling of frozen mouse retinal sections with purified sv2- <dig> antibodies repeatedly produced strong staining of endothelial cells of the inner retinal capillaries .
weaker but distinct labelling was also observed in the synapses of the outer plexiform layer and the photoreceptor outer segments.
preadsorption of sv2- <dig> with gst-abcc5_sv <dig> fusion protein completely abolished the immunostaining in the mouse retina  indicating that the labelling is specific.
in the rpe, a tissue with high abcc5_sv <dig> transcription , sv2- <dig> antibodies produced markedly staining of the apical and basolateral surfaces with exclusion of the rpe microvilli .
discussion
here we characterize three short abcc <dig> splice variants which consist of sequences corresponding to the first  <dig> exons of the abcc <dig> gene but revealing distinct 3' ends.
the isoforms are generated by the inclusion of one or two novel exons within intron  <dig> of the abcc <dig> 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 abcc <dig> transcript and the shorter abcc <dig> splice variants are present at varying levels in a number of tissues while all are predominantly expressed in the human retina.
although abcc <dig> mrna expression has repeatedly been found in neurons of the cns  <cit> , this is the first report of abcc <dig> being expressed in the neurosensory retina.
abcc <dig> can therefore be added to the list of abundant abc transporters with a function in the eye which for example includes abca <dig>  the gene underlying stargardt's disease  <cit>  and abcc <dig>  the gene implicated in pseudoxanthoma elasticum  <cit> .
so far, a role for abcc <dig> 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  <cit> .
est-based studies indicated a particularly high level of alternative splicing in neuronal tissues including the retina  <cit> .
the high fraction of splice variants among retinal cdnas are reflected in numerous reports of alternatively spliced retinal genes .
moreover, retina-specific mrna processing has been reported for genes with a broader tissue distribution  <cit> .
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  <cit> .
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 abcc <dig> gene in the retina demonstrate that one isoform, abcc5_sv <dig>  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   <cit> .
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   <cit> .
our data obtained from nmd inactivation and confirmed by rna interference show that the expression level of full length abcc <dig> transcript is influenced by the presence of alternatively spliced abcc <dig> isoforms, in particular abcc5_sv <dig>  rust therefore may play a role in abcc <dig> gene regulation.
alternative splicing of genes encoding abc transporters has previously been reported  <cit> .
noticeably, alternative splicing of two evolutionarily conserved ptc-containing exons of the abcc <dig> gene produces mrnas that are degraded by nmd  <cit> .
regulation of abcc <dig> gene expression is thought to be accomplished by facilitating the re-initiation of translation.
as a consequence shorter abcc <dig> proteins lacking a potentially important amino-terminal linker domain would be generated.
the newly identified abcc <dig> 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_sv <dig> 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 abcc <dig> gene may be a mechanism to increase protein diversity.
a polyclonal antibody directed against the c-terminus of the abcc <dig> transporter has been widely used to determine the tissue distribution of abcc <dig> in several organs.
among other cell types, this antibody strongly stains capillary endothelial cells in the genitourinary tract  <cit> , in the heart  <cit>  and in the brain  <cit> .
in brain, a contribution of abc transporters including abcc <dig> to the blood-brain barrier is discussed  <cit> .
similarly, the abcc5_sv <dig> isoform could play a role in the inner and outer blood-retinal barrier function possibly by controlling abcc <dig> transporter activity.
CONCLUSIONS
here we show that alternative splicing plays a role in the regulation of abcc <dig> gene expression via nmd-related mechanisms.
in addition, we present evidence that at least one of the abcc <dig> 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 abcc <dig> and its regulatory as well as protein-encoding isoforms in the retina.
this may also shed light onto a possible contribution of abcc <dig> to retinal disease.

 atp-binding cassette 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 mammalian abc transporters have been classified into seven subfamilies with the closely related multidrug resistance proteins grouped together in the c branch of abc proteins. abcc 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 abcc as a cellular export pump for numerous compounds including cgmp, nucleoside monophosphate analogs, heavy metal compounds and fluorochromes. abcc5-transfected cells were also reported to exhibit resistance to anticancer and antiviral drugs. the affinity of abcc to its substrates, however, has generally been low. this suggests that the biological significance of abcc 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 abcc gene is widely transcribed among human tissues with highest levels in heart, brain, skeletal muscle, kidney and testis. multiple mrna species for various abcc family members have been described including the abcc locus. sequencing of a single cdna clone from a human lung cancer cell line identified a splice variant of abcc formed by the alternative usage of a cryptic donor splice site upstream of exon. this so-called short type of multidrug resistance protein homologue translates into an n-terminally truncated version of abcc and shows a similar expression pattern as the full length abcc transcript. the physiological relevance of the rare smrp transcript is not known. in this study we have characterized three novel isoforms of the abcc gene generated by alternative splicing of newly identified exons within intron of the abcc gene. the various abcc 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 abcc mrna may provide an elegant mechanism to achieve a tissue-dependant regulation of abcc gene expression.  cloning of three novel abcc isoforms large-scale sequencing of a human retinal cdna library revealed two small cdna clones, 6d2f and a06-ret, located within intron of the abcc gene. subsequent blastn database queries revealed the retinal sequences to correspond to the 3' end of three overlapping full length cdna clones isolated from cancer cells and placenta. after assembly, the human mrna sequences from genbank comprise the first five exons of the abcc gene spliced to two alternative exons within intron of the abcc gene, named exon 5a and 5b. to further explore the existence of novel abcc splice variants in retina, we performed rt-pcr on human retinal rna with primers located in exon and exon 5b. three distinct pcr products of and base pairs were repeatedly amplified. direct sequencing of the pcr products showed that the two smaller fragments differ by the inclusion of exon 5a. in addition, the usage of an alternative splice donor sequence produces a larger exon 5a giving rise to the bp fragment. rt-pcr with primers located in exon and exon amplified a single bp fragment lacking exon 5a, 5aa or 5b indicating that the newly identified exonic sequences represent the respective 3'-ends of novel abcc splice variants abcc5_sv, abcc5_sv and abcc5_sv. this is confirmed by the presence of a potential consensus polyadenylation signal, aataaa, within bases of the 3' end of exon 5b. translation of the putative abcc5_sv abcc5_sv and abcc5_sv mrna sequences revealed stop codons in exon 5aa, exon 5a and exon 5b, respectively. this should lead to putative truncated abcc proteins which share the n-terminal amino acids with the full length abcc transporter and terminate in short distinct c-termini of, and amino acids in length. nucleotide homology searches using the novel abcc exons as templates showed that the sequence and the flanking splice junctions of exon 5a are highly conserved between mammalian species. analysis of est sequences from different species confirmed that exon 5a is commonly spliced to exon whereas each species uses different donor splice sites to join exon 5a to downstream sequences within intron of the abcc gene. thus, sequences overlapping but not identical to human exon 5b are frequently included in alternative transcripts. the presence of abcc splice variants with additional exons within intron of the abcc gene appears to be a common feature among mammalian species possibly indicating a conserved function of these molecules. expression analysis qualitative rt-pcr analysis revealed varying transcription levels of the novel splice variants in human retina with an increase of expression from abcc5_sv to abcc5_sv. the tissue distribution of the novel abcc splice variants was studied by virtual northern blot analysis with a radiolabeled probe representing exon 5b. a single strong band of approximately kb was detected exclusively in retina but not in bladder, heart, lung, skeletal muscle and rpe. to further investigate the relative expression of the splice variants, quantitative real-time rt-pcr with primer pairs specific for each of the abcc transcripts was performed with rna extracted from ten human tissues including samples from three distinct brain areas. an overall abundant expression of abcc and the shorter abcc isoforms was observed in human neural retina. in addition, full length abcc is expressed at moderate levels in several tissues including brain, rpe, bladder and heart. a relatively high expression in rpe, bladder and stomach was found for abcc5_sv whereas abcc5_sv and abcc5_sv are only weakly present in nonneuronal tissues. moderate expression of abcc5_sv and abcc5_sv was detected in cerebellum. taken together, our data show that each tissue analyzed expresses the various abcc transcripts but at distinct levels suggesting a complex regulation of gene expression. nonsense mediated decay of abcc isoforms a stop codon which resides over 50– nucleotides upstream from a downstream exon/exon junction is predicted to be recognized by the translational machinery as a premature termination codon and thus should be targeted by nonsense mediated mrna decay. splice variant abcc5_sv contains a stop codon bp upstream of the exon 5aa/5b splice junction fulfilling the criteria for a ptc. to determine whether the abcc5_sv mrna is subject to nmd we inactivated the nmd surveillance mechanism by the addition of translation inhibitors puromycin, or alternatively anisomycin, to human cell lines arpe- and y which were shown before to express abcc abcc5_sv abcc5_sv and abcc5_sv. by qrt-pcr, puromycin treatment drastically increased the relative expression of abcc5_sv mrna by almost 3-fold in the arpe- cell line and by more than 2-fold in the y cell line. interestingly, in both cell lines the expression of the abcc transcript decreased by a factor of approximately two. in contrast, the relative expression levels of abcc5_sv and abcc5_sv remained unaffected by the addition of the translation inhibitors. the concentration of the mrnas largely returned to pre-treatment levels after protein synthesis was restored by removing puromycin from the media. similar to the treatment with puromycin, application of anisomycin lead to a 2- and 4-fold increase of abcc5_sv mrna in y and arpe- cells, respectively, whereas the levels of the remaining abcc transcript variants were stable or decreased. together, these data demonstrate that inhibition of cellular protein synthesis leads to a selective stabilization of the abcc5_sv mrna indicating that this transcript is regulated by nmd. in addition, elevated levels of abcc5_sv mrna appear to regulate abcc gene expression without exerting any effects on the remaining short abcc isoforms. silencing of the novel abcc isoforms by rna interference to further investigate whether the expression of the various abcc transcripts may be co-regulated, abcc5_sv abcc5_sv and abcc5_sv were silenced by rna interference. consequently, treatment of arpe- cells with an sirna probe targeted to exon 5b decreased the expression of abcc5_sv abcc5_sv and abcc5_sv up to 80% while the expression of abcc increased -fold over time. a similar rise in abcc expression was observed when abcc5_sv and abcc5_sv expression was selectively suppressed by sirna.. the level of abcc5_sv essentially remained unaffected. sirna probes to specifically target abcc5_sv without affecting the other two isoforms were designed, but consistently failed to reveal a measurable knock down effect of abcc5_sv expression. from our results we conclude that abcc mrna expression is differentially regulated by the presence of one or more short abcc splice variants, most likely by abcc5_sv and/or abcc5_sv localization of abcc5_sv in the retina the most c-terminal amino acid peptide specifically encoded by the abcc5_sv isoform appears highly conserved in a number of mammalian species suggesting a functional constraint on this sequence. to investigate whether abcc5_sv indeed is expressed as a short abcc protein isoform, we generated polyclonal antibodies directed against its unique amino acid terminus. immunofluorescence labeling of frozen mouse retinal sections with purified sv2- antibodies repeatedly produced strong staining of endothelial cells of the inner retinal capillaries. weaker but distinct labelling was also observed in the synapses of the outer plexiform layer and the photoreceptor outer segments. preadsorption of sv2- with gst-abcc5_sv fusion protein completely abolished the immunostaining in the mouse retina indicating that the labelling is specific. in the rpe, a tissue with high abcc5_sv transcription, sv2- antibodies produced markedly staining of the apical and basolateral surfaces with exclusion of the rpe microvilli. discussion here we characterize three short abcc splice variants which consist of sequences corresponding to the first exons of the abcc gene but revealing distinct 3' ends. the isoforms are generated by the inclusion of one or two novel exons within intron of the abcc 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 abcc transcript and the shorter abcc splice variants are present at varying levels in a number of tissues while all are predominantly expressed in the human retina. although abcc mrna expression has repeatedly been found in neurons of the cns, this is the first report of abcc being expressed in the neurosensory retina. abcc can therefore be added to the list of abundant abc transporters with a function in the eye which for example includes abca the gene underlying stargardt's disease and abcc the gene implicated in pseudoxanthoma elasticum. so far, a role for abcc 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. 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. moreover, retina-specific mrna processing has been reported for genes with a broader tissue distribution. 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 abcc gene in the retina demonstrate that one isoform, abcc5_sv 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. 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. our data obtained from nmd inactivation and confirmed by rna interference show that the expression level of full length abcc transcript is influenced by the presence of alternatively spliced abcc isoforms, in particular abcc5_sv rust therefore may play a role in abcc gene regulation. alternative splicing of genes encoding abc transporters has previously been reported. noticeably, alternative splicing of two evolutionarily conserved ptc-containing exons of the abcc gene produces mrnas that are degraded by nmd. regulation of abcc gene expression is thought to be accomplished by facilitating the re-initiation of translation. as a consequence shorter abcc proteins lacking a potentially important amino-terminal linker domain would be generated. the newly identified abcc 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_sv 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 abcc gene may be a mechanism to increase protein diversity. a polyclonal antibody directed against the c-terminus of the abcc transporter has been widely used to determine the tissue distribution of abcc 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 abcc to the blood-brain barrier is discussed. similarly, the abcc5_sv isoform could play a role in the inner and outer blood-retinal barrier function possibly by controlling abcc transporter activity.  here we show that alternative splicing plays a role in the regulation of abcc gene expression via nmd-related mechanisms. in addition, we present evidence that at least one of the abcc 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 abcc and its regulatory as well as protein-encoding isoforms in the retina. this may also shed light onto a possible contribution of abcc to retinal disease. 
two additional exons within intron of the abcc gene were identified; one of the exons exhibits alternative donor splice sites. finally, we show that the evolutionarily conserved abcc5_sv transcript is translated into a protein abundantly present in endothelial cells of inner retinal blood vessels and along rpe membranes. abcc and its novel isoforms are abundantly expressed in the human retina.
 the abcc gene encodes an organic anion pump of the atp-binding cassette transporter family, subclass c. the exact physiological function of abcc however is not known. here, we have isolated three novel abcc splice variants and characterized their role in the regulation of abcc gene expression.  two additional exons within intron of the abcc gene were identified; one of the exons exhibits alternative donor splice sites. differential usage of these exons generates three short abcc transcripts named abcc5_sv abcc5_sv and abcc5_sv the variants share the first five exons with the abcc gene but differ in their 3' sequences. abcc and its novel isoforms are abundantly expressed in the human retina. splice variant abcc5_sv and abcc5_sv contain premature stop codons. while inhibition of nonsense-mediated mrna decay selectively stabilized abcc5_sv but not abcc5_sv the amount of full length abcc mrna was simultaneously reduced. a negative regulatory effect on full length abcc expression was also observed when the abcc isoforms were silenced with sirna duplexes. finally, we show that the evolutionarily conserved abcc5_sv 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 abcc gene has functional consequences by modulating abcc gene expression. in addition, at least one abcc splice variant is protein-coding and produces a truncated abcc protein isoform with thus far unknown functional properties in the retina. 