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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

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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 6

Quantitative rescue of NMD-sensitive mutant mRNA alleles with cycloheximide and emetine treatment.

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Quantitative rescue of NMD-sensitive mutant mRNA alleles with cyclohexim...
Representative images of agarose gel and capillary electrophoresis of semiquantitative RT-PCR products from mutant PRPF31 transcripts are shown (for emetine only). For each cell line, PCR products were always run on the same agarose gel, but they were not contiguous. Data shown summarize measurements of 5 PCR replicates from 3 (emetine) or 4 (cycloheximide) treatments on independent cultures for each cell line. Cell line identification codes are shown on the left. The term ctrl refers to semiquantitative RT-PCR from a control cell line (GM104848). Throughout, the symbols + and – indicate the presence or absence of NMD inhibitors in the cell culture medium. The terms mut and wt correspond to PCR products from plasmids containing the mutant and wild-type alleles, respectively. Electrophoretograms were normalized to the height of the wild-type peak and aligned automatically. Black and red curves indicate the absence or the presence of emetine in the culture medium, respectively, and asterisks highlight PCR products derived from the mutant mRNA form. Treatment with either cycloheximide (blue bars) or emetine (red bars) partly restored the expression of NMD-sensitive mutant alleles and had no effect on NMD-insensitive mutant alleles. For cell lines AG0293, AG0305, and AG0307, bars show only the change in the NMD-sensitive allele (short form). Data presented as bar graphs are the combination of both densitometry values of agarose gel bands (ImageJ software) and the integration of peak from capillary electrophoresis (Biocalculator software).

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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