Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression

Background 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. Results 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. Conclusion 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

Evolution and functional divergence of the anoctamin family of membrane proteins

Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family

Mutations in the Genes for Interphotoreceptor Matrix Proteoglycans, IMPG1 and IMPG2, in Patients with Vitelliform Macular Lesions

A significant portion of patients diagnosed with vitelliform macular dystrophy (VMD) do not carry causative mutations in the classic VMD genes BEST1 or PRPH2. We therefore performed a mutational screen in a cohort of 106 BEST1/PRPH2-negative VMD patients in two genes encoding secreted interphotoreceptor matrix proteoglycans-1 and -2 (IMPG1 and IMPG2). We identified two novel mutations in IMPG1 in two simplex VMD cases with disease onset in their early childhood, a heterozygous p.(Leu238Pro) missense mutation and a homozygous c.807 + 5G > A splice site mutation. The latter induced partial skipping of exon 7 of IMPG1 in an in vitro splicing assay. Furthermore, we found heterozygous mutations including three stop [p.(Glu226*), p.(Ser522*), p.(Gln856*)] and five missense mutations [p.(Ala243Pro), p.(Gly1008Asp), p.(Phe1016Ser), p.(Tyr1042Cys), p.(Cys1077Phe)] in the IMPG2 gene, one of them, p.(Cys1077Phe), previously associated with VMD. Asymptomatic carriers of the p.(Ala243Pro) and p.(Cys1077Phe) mutations show subtle foveal irregularities that could characterize a subclinical stage of disease. Taken together, our results provide further evidence for an involvement of dominant and recessive mutations in IMPG1 and IMPG2 in VMD pathology. There is a remarkable similarity in the clinical appearance of mutation carriers, presenting with bilateral, central, dome-shaped foveal accumulation of yellowish material with preserved integrity of the retinal pigment epithelium (RPE). Clinical symptoms tend to be more severe for IMPG1 mutations

Cellular localization of the MPP4 protein in the mammalian retina

PURPOSE. Membrane protein, palmitoylated (MPP)-4 is a novel retina-specific member of the p55-like subfamily of membraneassociated guanylate kinases (MAGUKs). MAGUKs are known to act as scaffolding molecules for multiprotein complexes at specialized regions of the plasma membrane. The goal of this study was to characterize the MPP4 protein and to determine its location in the mammalian retina. METHODS. RT-PCR and 5 � and 3 � rapid amplification of cDNA ends (RACE) techniques were used to isolate and sequence the full-length bovine MPP4 cDNA. Polyclonal antisera against the bovine MPP4 protein were generated in rabbits immunized with synthetic peptides. Affinity-purified anti-MPP4 antibodies were used to investigate the properties and distribution of MPP4 in retina and transfected 293-Ebna cells by Western blot analysis and immunofluorescence microscopy

Cloning and characterization of WDR17, a novel WD repeat-containing gene on chromosome 4q34

8 páginas, 3 figuras, 1 tabla.-- et al.As part of our project to generate a catalogue of genes with potential relevance to human retinal disease, we have cloned a transcript abundantly expressed in the human retina and testis. Analysis of the deduced 1322 amino acid protein sequence demonstrates that it encodes a novel WD repeat protein, termed WDR17. The N-terminal moiety of the WDR17 protein is predicted to consist of at least 12 conserved WD repeats that likely adopts a β-propeller-like structure. Homology searches with the C-terminal region revealed no similarity to known or hypothetical proteins. However, putative orthologous ESTs with 82–91% identity to the human cDNA were found in several mammalian species including rodents, pig and cattle, suggesting that WDR17 represents an evolutionarily novel subtype of WD repeat proteins with unique function(s) in higher eukaryotes. Temporal expression analysis in the murine eye showed that transcription of WDR17 begins prenatally, suggesting a functional role of the protein in the early stages of retinal development. Human WDR17 maps to the same chromosomal interval as the locus for autosomal recessive retinitis pigmentosa (RP29) on 4q34, making it a candidate for this disease gene. Sequencing of the entire coding region of WDR17 in an affected patient of the original RP29 pedigree has not revealed any disease-causing sequence variations likely excluding WDR17 as the gene underlying RP29.This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) under 01KW9921/0.Peer reviewe

Molecular dissection of TIMP3 mutation S156C associated with Sorsby fundus dystrophy

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular degeneration of late onset. A key feature of the disease is the thickening of Bruch's membrane, an ECM structure located between the RPE and the choroid. SFD is caused by mutations in the gene encoding the ECM-associated tissue inhibitor of metalloproteases-3 (TIMP3). We have recently generated two Timp3 gene-targeted mouse lines, one deficient for the murine gene (Timp3−/−) and one carrying an SFD-related S156C mutation. Based on extracts and cell cultures derived from tissues of these animals we now evaluated TIMP3 functionality and its contribution to SFD. We show that the activity levels of TIMP3 target proteases including TACE, ADAMTS4/5 and aggrecan-cleaving MMPs are similar in Timp3S156/+ and Timp3S156C/S156C mice when compared to controls. In Timp3−/− mice, a significant enhancement of enzyme activity was observed for TACE but not for ADAMTS4/5 and MMPs indicating a compensatory effect of other inhibitors regulating the latter two groups of proteases. Fibrin bead assays show that angiogenesis in Timp3S156/+ and Timp3S156C/S156C mice is not altered whereas increased formation of capillary tubes was observed in Timp3−/− animals over controls. Rescue experiments using recombinant proteins demonstrate that the inhibitory activities of TIMP3 towards TACE and aggrecan-cleaving MMPs as well as the anti-angiogenic properties of TIMP3 are not impaired by SFD mutation S156C. We finally demonstrate that wild-type and S156C-TIMP3 proteins block the binding of VEGF to its receptor VEGFR2 to a similar extent. Taken together, this study shows that S156C-TIMP3 retains its known functional properties suggesting that causes other than an imbalance in protease or angiogenic activities represent the primary molecular defect underlying SFD

Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression-3

<p><b>Copyright information:</b></p><p>Taken from "Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression"</p><p>http://www.biomedcentral.com/1471-2199/8/42</p><p>BMC Molecular Biology 2007;8():42-42.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890297.</p><p></p>eting ABCC5_SV1 and ABCC5_SV2. ARPE-19 transfected with a nonsense siRNA was used as a negative control. Total RNA isolated 8 h, 24 h and 48 h after transfection was used for qRT-PCR analysis. The respective expression values are shown by specific symbols. The expression level of cells transfected with nonsense siRNA was set at 100%. Bar graphs represent standard deviations of reactions performed in triplicate

Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression-0

<p><b>Copyright information:</b></p><p>Taken from "Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression"</p><p>http://www.biomedcentral.com/1471-2199/8/42</p><p>BMC Molecular Biology 2007;8():42-42.</p><p>Published online 23 May 2007</p><p>PMCID:PMC1890297.</p><p></p> shown as open boxes, the retinal cDNA fragments as solid boxes. The mRNArepresents a cDNA assembled from three human mRNA sequences deposited in GenBank [, and ]. The locations of primers used to PCR amplify ABCC5 cDNA fragments and the start and stop codon of full length ABCC5 are indicated by solid triangles above. Numbers refer to the respective exon number. The vertical line in exon 5Aa indicates an alternative splice donor site (B) Differential splicing of exons 5A, 5Aa and 5B generates three ABCC5 isoforms in the human retina, termed ABCC5_SV1, ABCC5_SV2 and ABCC5_SV3. RNA from human retina was RT-PCR amplified with primer pair F1/R3