Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay
Thomas Rio Frio, … , Jacques S. Beckmann, Carlo Rivolta
Thomas Rio Frio, … , Jacques S. Beckmann, Carlo Rivolta
Published March 3, 2008
Citation Information: J Clin Invest. 2008;118(4):1519-1531. https://doi.org/10.1172/JCI34211.
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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 2

Capillary electrophoresis of semiquantitative RT-PCRs spanning 6 different PRPF31 mutations.

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Capillary electrophoresis of semiquantitative RT-PCRs spanning 6 differe...
We analyzed unprocessed PCR products obtained with 6 different primer pairs, specific for each mutation, that could simultaneously amplify the wild-type and the mutant mRNA (cDNA) in the same reaction. The single curves shown are representative of 5 replicates from each of 3 independent cultures. The x axis indicates the approximate size (in bp) of the DNA fragments, whereas the y axis shows the amount of PCR product normalized to the peak height of the wild-type allele. PCR products originating from the mutant alleles are indicated by asterisks. For c.1115_1125del, the peak at ~197 bp is the short form produced by the skipping of exon 11, whereas the peak at ~260 bp is the NMD-insensitive long-form mRNA allele containing the 11-bp deletion. For the c.856-2A>G mutation, the red curve corresponds to cell line 12688, whereas the blue curve corresponds to cell line 13190. For the c.1115_1125del mutation, the PCR products from cell lines AG0293, AG0305, and AG0307 are indicated by the green, red, and blue curves, respectively.