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SE Wilkie, E Deery, EN Vithana, C Chakarova, RJ Newbold, MJ Warren, SS Bhattacharya, DM Hunt; Functional Characterisation of Missense Mutations in the Splicing Factor Gene PRPF31 Underlying Autosomal Dominant Retinitis Pigmentosa (RP11) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2724.
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Purpose:A series of mutations that include missense substitutions, deletions and insertions, has been identified in the putative splicing factor gene PRPF31 in RP11 patients. This study aims to characterise the functional defects arising from some of these missense mutations. Methods:Wild type and mutant human PRPF31 proteins were expressed as His-tagged fusion proteins in mammalian cells. Relative expression levels of the recombinant proteins in 293T cells were assessed by Western analysis using anti-His antibodies. The profiles of total cell protein from cells expressing wild type and mutant PRPF31 were studied by SDS-PAGE with Coomassie staining. Localisation of the proteins expressed in COS7 cells was studied by immunoprobing of cells grown on glass coverslips and observation using confocal fluorescence microscopy. Results:Expression of wild type PRPF31 and of three missense mutants was not toxic to host 293T cells and no gross differences in the profiles of total cell protein were observed. However, the mutant proteins accumulated to higher levels than the wild type.Wild type PRPF31 expressed in COS7 cells localised exclusively to cell nuclei. A mutant in which residues 351-364 had been deleted failed to translocate to the nucleus, confirming this as the location of the nuclear localisation sequence. Conclusion:Wild type PRPF31 is confirmed as a nuclear protein, consistent with its presumed function as an RNA splicing factor. Our results indicate no immediate deleterious effect of mutant PRPF31 on 293T cells, even when expressed at supra-physiological levels, although the increased accumulation of the mutant protein suggests that it is turned over more slowly than wild type. We are now developing an in vivo functional assay to probe for the presence of unspliced transcripts from cells expressing mutant PRPF31.
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