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S.E. Wilkie, K.J. Morris, S.S. Bhattacharya, M.J. Warren, D.M. Hunt; A Study of the Nuclear Trafficking of the Splicing Factor Protein PRPF31 Linked to Autosomal Dominant Retinitis Pigmentosa (ADRP) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5167.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study the mechanism of importation into the nucleus of the splicing factor protein PRPF31 and assess the impact of two mutations, A194E and A216P, linked to the retinal disease ADRP. Methods: His–tagged wild type and mutant PRPF31 were purified from bacterial cell extracts. Pull–down assays were used to study the interaction of these His–tagged PRPF31 proteins with GST–tagged importins. Fluorescence recovery after photobleaching (FRAP) was used to estimate the real time rate of movement of EGFP–tagged PRPF31 into the nuclei of transfected COS7 cells. Results: PRPF31 interacts with importin ß1 for translocation to the nucleus, with no requirement for importin α1. The A194E and A216P mutations had no apparent affect on this interaction. FRAP analysis indicated that the movement of EGFP–tagged PRPF31 follows a two–component recovery process, a fast component with τ∼6 sec and a slow component with τ∼80 sec. The mutations affected neither component. However, cells transfected with the EGFP–tagged mutant constructs indicated the formation of aggresomes within the nuclei. Conclusions: The A194E and A216Pmutations have no effect on the importation of PRPF31 into the nuclei of COS7 cells, but reduced protein solubility results in aggresome formation. Whilst aggresomes in the nuclei of rod photoreceptors could contribute to the pathology in ADRP patients, a more serious problem may be the concomitant insufficiency of splicing activity in cells with a very high metabolic demand. Our failure to find any dominant negative effect of these mutations renders the disease an excellent candidate for treatment by gene therapy targeted to rod photoreceptors.
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