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D. W. Anderson, C. F. Chakarova, M. Dawiczyk, S. S. Bhattacharya, D. S. Papermaster; Mutations in PRP3 Cause Retinal Degeneration in a Transgenic X. laevis Model of Human ADRP. Invest. Ophthalmol. Vis. Sci. 2007;48(13):580.
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Two mutations in the Human PRP3 gene have been linked to an autosomal dominant form of retinitis pigmentosa (adRP, RP18, Chakarova et al., 2002). PRP3 is a 683 amino acid protein involved in pre-mRNA splicing in association with the U4/U6 snRNP. The mutations occur in adjacent codons resulting in amino acid changes P493S and T494M. These amino acids are 100% conserved in species from yeast to mammals. We expressed the human PRP3 cDNA (hPRP3) and its associated mutations in transgenic X. laevis tadpoles to determine if a retinal degeneration could be induced. The X. laevis PRP3 cDNA (xPRP3) was obtained to ensure that the observed effects were the consequence of the mutation and not of species variations in the proteins.
The xPRP3 cDNA was obtained by RT-PCR of retinal mRNA; mutations were created by PCR and confirmed by sequencing. The hPRP3 and xPRP3 proteins were expressed as GFP fusion proteins under the control of the Xenopus opsin promotor (Moritz et al., 1999) with GFP on the N-termini. Transgenic tadpoles were fixed in 4% paraformaldehyde and the retinas visualized by confocal microscopy of frozen sections.
The xPRP3 cDNA encodes a 681 amino acid protein that is 86% identical to the human protein and 95% similar. The equivalent of the human mutations in the frog are P491S and T492M. Expression of the GFP-hPRP3 fusion proteins resulted in nuclear localization with a punctate appearance. Often a large spot was observed with multiple smaller puncta. The hPRP3-P493S mutant resulted in a degeneration of rods from the central retina in some tadpoles as early as 14 days post-fertilization (dpf). The hPRP3-T494M mutation did not result in a dramatic degenerative phenotype in the animals examined to date. Expression of the GFP-xPRP3-P491S fusion protein resulted in nuclear localization with a punctate pattern like that seen with GFP-hPRP3 fusion proteins. There were no obvious signs of degeneration in 14 dpf retinas, but two out of four 21 dpf retinas displayed a loss of rods from the central retina. There were many more GFP positive cells in the periphery of the affected retinas than in those that appeared normal.
Mutations in both hPRP3 and xPRP3 can induce a degeneration of rods in transgenic X. laevis retinas. This supports the interpretation that the mutations in the human pedigrees can cause adRP. It appears there may be a threshold level of the mutant protein required to cause rod death. More animals need to be studied to identify the mechanism of retinal degeneration.
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