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 4

Differential protein abundance and absence of truncated PRPF31 proteins in cell lines derived from patients.

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Differential protein abundance and absence of truncated PRPF31 proteins ...
(A) Quantitative SDS-PAGE (10%) of total protein extracts from various patient cell lines and controls. Simultaneous infrared detection of proteins was achieved with a highly specific N-terminal anti-PRPF31 antibody (green) and a β-actin control (red). The white dotted boxes indicate the expected position of mutant proteins derived from NMD-sensitive alleles, if they were present. Protein size cannot be predicted from the mutation in cell line 14523; however, it is likely to lack the N-terminal epitope for detection with this antibody. (B) Original magnification of another quantitative, high-resolution SDS-PAGE (15%) using protein extracts from a cell line carrying the c.1115_1125del mutation (AG0307) and from an unaffected control (AG0309). A high-percentage acrylamide gel, run over a longer time, was used to resolve the small size difference between PRPF31 proteins derived from the wild-type and the mutant long-form mRNA. However, only the full-length (wild-type) PRPF31 form was visible. (C) Quantification of PRPF31 protein abundance from cell lines derived from patients using a combination of 4 independent gels, including the one depicted in A, and normalized to the abundance of β-actin. Black bars, control cell lines; white bars, cell lines from patients with PRPF31 mutations.