Abstract
Purpose: :
The observation of partial disease penetrance in families carrying mutations in PRPF31 has previously suggested that disease phenotype results from an insufficiency of functional PRPF31. However, recent observation of mild clinical symptoms in so–called ‘asymptomatic’ carriers of the disease suggests a dominant negative effect of the mutations cannot be ruled out. This work aims to develop a splicing assay to detect any dominant effect of two pathogenic PRPF31 mutations (A194E and A216P) and to further investigate the functional effects of these mutations.
Methods: :
Splicing assays were conducted in a human cell line (293T) using constructs co–expressing PRPF31 (WT or mutant) together with a splicing template comprising full length or partial rhodopsin gDNA (RHO). The interaction between PRPF31 and its binding partner PRPF6 was studied by means of pull down assays using His–tagged PRPF31 (WT and mutant) and GST–tagged PRPF6.
Results: :
Removal of intron 3 from the RHO splicing template was inefficient with only ∼45% of RHO template correctly spliced. This was unaffected by co–expression of WT PRPF31 but co–expression of the two PRPF31 mutants reduced the proportion of spliced product to ∼25%. The pull–down assays showed that the 3’ terminal domain of PRPF6 (TPRs 11–18) interacts with PRPF31.Early results indicate that the two PRPF31 mutations have differential effects on the strength of binding, with the A194E mutant binding more strongly than WT.
Conclusions: :
The splicing assay indicates that both mutations have a dominant negative effect on splicing of RHO intron 3. At least in the case of the A194E mutant, this could derive from an increased interaction with PRPF6, which could interfere with the normal dissociation of snRNPs U4/U6 and U5 from the spliceosome in the regeneration part of the splicing cycle.
Keywords: proteins encoded by disease genes • retina: distal (photoreceptors, horizontal cells, bipolar cells) • photoreceptors