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V. Marigo, C. Spampanato, A. Comitato, D. Sanges; Functional studies of PRP3 pre–RNA splicing factor mutated in patients affected by Retinitis Pigmentosa . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3611.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Many genes mutated in retinitis pigmentosa (RP) are components of the phototransduction cascade. However, in the last 2 years, genes encoding pre–RNA splicing factors were found mutated in three forms of autosomal dominant RP (adRP, RP11, RP13 and RP18). The slicing factors involved in adRP are PRPF31, PRP3, PRPF8. The link between mutant splicing factors and retinal degeneration is completely obscure. Methods: RNA in situ hybridization studies for the analysis of the expression pattern of PRP3 in embryonic and adult eyes. Subcellular localization of the protein using transfection experiment. Subcellular localization of the mutant forms of Prp3 as found in patients. Results: We analyzed expression of the Prp3 gene in the developing and adult murine eye by RNA in situ hybridization. We found that Prp3 transcripts are ubiquitously distributed but higher amounts could be detected in the central nervous system and neural retina. Expression does not seem to be strictly correlated with the photoreceptor cell layer. Using in vitro transfection in HeLa, NIH3T3 and Cos7 cells, Prp3 localizes into the nuclei. Using similar approaches we found the two different mutations (P493S and T494M) have similar intracellular distribution as found for the wild–type protein. We then wanted to study Prp3 protein distribution compared to splicing proteins. Co–localization analysis of transfected cells using antibodies recognizing a protein of the splicing complex suggests that Prp3 only partially co–localizes with the spliceosome. Conclusions: We analyzed Prp3 transcript distribution in vivo and found no direct correlation with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. Co–localization with splicing factors confirms that the protein is part of the spliceosome. However the protein shows a more complex subcellular distribution suggesting additional roles of this protein. Finally, mutant Prp3 is translocated into the nucleus like the wild type, ruling out the possibility of mis–folding and degradation of the mutant protein.
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