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C.F. Chakarova, E.N. Vithana, S. Wilkie, D.M. Hunt, S.S. Bhattacharya; Functional Studies of Mutations in HPRP3 Associated with Autosomal Dominant Retinitis Pigmentosa . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2838.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study the functional consequences of two mutations, P493S and T494M, in splicing factor gene HPRP3 linked to autosomal dominant retinitis pigmentosa (RP18). Methods: Retinal cDNA was used for full length amplification of two known splicing factor genes HPRP3 and HPRP4 and their interacting component Cyclophylin H (CycH). The fragments were cloned into the expression vector pTriEx-1 with C-terminal His.tag. The resulting construct was used to transfect COS7 cells and human cell line HEK 293T. Results: Protein extracts from transfected 293T cells were examined by Western blot analysis where the expression of the proteins HPRP3, HPRP4 and CycH has been demonstrated using specific antibodies. COS7 cells were used in confocal microscopy experiments for sub-cellular localisation of the wild type (WT) and mutant HPRP3 proteins. Preliminary results show that WT HPRP3 and both mutants (P493S and T494M) localise in the nucleus of the cells. Co-immunoprecipitation studies using HPRP4 and Cyclophylin H are underway to evaluate the impact of mutations on protein interactions. Conclusions: Our data suggests that both mutant and WT HPRP3 localises in the nucleus of the cells. However, loss of photoreceptors in RP patients may be due to a failure in protein interactions between HPRP3 and its molecular partners – HPRP4 and CycH affecting RNA splicing.
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