Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay
Thomas Rio Frio, Nicholas M. Wade, Adriana Ransijn, Eliot L. Berson, Jacques S. Beckmann, Carlo Rivolta
Thomas Rio Frio, Nicholas M. Wade, Adriana Ransijn, Eliot L. Berson, Jacques S. Beckmann, Carlo Rivolta
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Research Article Genetics

Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay

Abstract

Dominant mutations in the gene encoding the mRNA splicing factor PRPF31 cause retinitis pigmentosa, a hereditary form of retinal degeneration. Most of these mutations are characterized by DNA changes that lead to premature termination codons. We investigated 6 different PRPF31 mutations, represented by single-base substitutions or microdeletions, in cell lines derived from 9 patients with dominant retinitis pigmentosa. Five of these mutations lead to premature termination codons, and 1 leads to the skipping of exon 2. Allele-specific measurement of PRPF31 transcripts revealed a strong reduction in the expression of mutant alleles. As a consequence, total PRPF31 protein abundance was decreased, and no truncated proteins were detected. Subnuclear localization of the full-length PRPF31 that was present remained unaffected. Blocking nonsense-mediated mRNA decay significantly restored the amount of mutant PRPF31 mRNA but did not restore the synthesis of mutant proteins, even in conjunction with inhibitors of protein degradation pathways. Our results indicate that most PRPF31 mutations ultimately result in null alleles through the activation of surveillance mechanisms that inactivate mutant mRNA and, possibly, proteins. Furthermore, these data provide compelling evidence that the pathogenic effect of PRPF31 mutations is likely due to haploinsufficiency rather than to gain of function.

Authors

Thomas Rio Frio, Nicholas M. Wade, Adriana Ransijn, Eliot L. Berson, Jacques S. Beckmann, Carlo Rivolta

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Figure 8

Inhibition of NMD and the lysosomal and proteasomal protein degradation pathways does not rescue the synthesis of mutant PRPF31 proteins.

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Inhibition of NMD and the lysosomal and proteasomal protein degradation ...
(A) The addition of wortmannin (WRT) markedly enhanced the stability of most mutant forms of mRNA as measured by semiquantitative RT-PCR, except for mRNA derived from the c.177+1delG mutation that skips the initiation codon. A similar and sometimes more pronounced rescue was observed in the presence of WRT along with the lysosome inhibitor chloroquine (CHQ) and the proteasome inhibitor MG132. Data summarize measurements of 5 PCR replicates from 3 independent cultures treated as shown for each of the 6 cell lines. (B) Quantitative SDS-PAGE of the corresponding protein extracts from the same cell preparations. WRT treatment alone did not rescue the expression of any mutant proteins, nor did the addition of lysosomal and proteasomal inhibitors. Sample 14523 can be used as a negative control because it lacks the N-terminal epitope needed for recognition with this antibody.