Abstract
Abstract: :
Purpose: ABCC5 is an ATP–binding cassette transporter acting as an ATP–dependent export pump for cGMP and thus represents a rapid elimination pathway for this universal signalling molecule. ABCC5 is widely expressed among tissues. In the present study, we have identified alternatively spliced exons of ABCC5 giving rise to three short isoforms abundantly expressed in the retina. We have further characterized these isoforms and have addressed their functional properties. Methods: Real–time quantitative RT–PCR in 21 different human tissues including retina was performed with isoform–specific oligonucleotide primers. RNA interference with siRNA duplexes and protein synthesis inhibition assays were done in the RPE cell line ARPE–19 and the retinoblastoma cell line Y79. Antibody SV2–304 was generated against an ABCC5 isoform specific recombinant protein. Results: By inhibiting protein synthesis, we demonstrate that splice variant SV1 is a target for nonsense mediated decay (NMD). In addition, selective silencing of the short ABCC5 isoforms shows an inverse correlation of SV1 expression with the transcript level of ABCC5. Splice variant SV2 encodes a 209 aa protein and was localized to the inner retina endothelial cells. Cellular expression of isoform SV3 is still unknown. Conclusions: The ubiquitous expression of ABCC5 may be subjected to tissue–specific transcriptional regulation by a mechanism involving alternative splicing of its pre–mRNA. Furthermore, the presence of a short ABCC5 protein isoform lacking the two membrane spanning domains and the two nucleotide binding domains indicates a novel role distinct from ABCC5.
Keywords: gene/expression • immunohistochemistry • transcription