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F. Fiocco, C. Maubaret, R. Roepman, F. P. M. Cremers, K. Matter, S. S. Bhattacharya; Investigation and Characterization of PRPF31 Interacting Proteins in Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):776. doi: https://doi.org/.
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Mutations in PRPF31 gene cause autosomal dominant retinitis pigmentosa 11 (RP11). Interestingly, mutations in this ubiquitously expressed gene do not lead to any other phenotypes. The aim is to identify retina-specific partners for PRPF31 to elucidate its function.
Potential partners of PRPF31 have been sought using the yeast two-hybrid system. Both a randomly primed bovine retina cDNA library and an oligo-dT primed human retina cDNA library were screened, using PRPF31 as a bait. RT-PCR was used to confirm the presence of the candidate partners in retina. Interaction between PRPF31 and putative partners has been investigated through co-immunoprecipitation studies on pig retinal lysates.
Three potential candidates have been isolated from the bovine retina library: MAGI3 (membrane-associated guanylate kinase with inverted orientation, 3), TRAK2 (trafficking kinesin-binding protein 2), and ZNF143 (zinc finger protein 143). RT-PCR analysis of the genes encoding these proteins proved their expression in the retina, although they are not retina-specific. However, amplification of additional bands may indicate that retina-specific isoforms exist. Pig retinal lysates were used for co-immunoprecipitation studies to validate the identified interactions.
MAGI3, TRAK2 and ZNF143 have been isolated as potential interacting partners for PRPF31 in retina. RT-PCR confirmed the expression of the MAGI3, TRAK2 and ZNF143 genes in human retina. Ongoing work includes further validation of the identified interactions by co-localisation and co-immunoprecipitations studies. In HEK293T cells by using the recombinant proteins, and by scrutinizing the native protein complexes in the retina using specific antibodies against the target proteins in co-immunoprecipitation assays and immunohistochemical analyses.
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