Abstract
Purpose::
Mutations in the pre-mRNA splicing factor PRPF31 (RP11) gene have been associated with autosomal dominant retinitis pigmentosa (RP). In this study, we are investigating the early molecular mechanisms that may identify a link between PRPF31 mutations, spliceosome function and the observed retinal specific phenotypes in RP.
Methods::
Immortalised lymphoblast cell lines were derived from six affected and three asymptomatic patients, encompassing six distinct RP11 mutations of which five cause the formation of premature termination codons (PTCs). Qualitative PRPF31 allelic expression for each cell line was measured in the presence and absence of the NMD inhibitor cycloheximide by RT-PCR, using primers specific to each patient mutation.
Results::
In all patient cell lines, the expression level of the mutant PRPF31 allele was significantly reduced or absent compared to the non-mutated allele. However, incubation in cycloheximide partially reversed this effect and restored some expression of the mutated allele in all five patient cell lines containing mutations resulting in PTCs. In cells with the remaining mutation, a deletion of the normal start site in exon 2, the mutated allele did not increase upon cycloheximide treatment.
Conclusions::
NMD appears to target and degrade mutated PRPF31 alleles in patients with dominant RP11 mutations that encode PTCs. Mutations that do not result in PTCs appear to be down-regulated by another mechanism unrelated to NMD, or at least using a mechanism unaffected by cycloheximide. This prevents the translation of potentially deleterious truncated PRPF31 proteins and suggests that dominant negative effects are unlikely to be responsible for the pathogenesis of retinitis pigmentosa. We are further characterising the role of NMD in the regulation of mutated PRPF31 alleles by specific RNAi inhibition of the essential NMD protein Upf1/RENT1. Using this system, we are in the process of further investigating the influence of NMD on PRPF31 protein abundance and sub-nuclear localisation in patient cells in comparison to unaffected controls.
Keywords: gene/expression • retinal degenerations: hereditary • mutations