CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance

Giulia Venturini, Anna M Rose, Amna Z Shah, Shomi S Bhattacharya, Carlo Rivolta, Giulia Venturini, Anna M Rose, Amna Z Shah, Shomi S Bhattacharya, Carlo Rivolta

Abstract

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. CNOT3 shows an opposite trend…
Figure 1. CNOT3 shows an opposite trend of expression with respect to that of PRPF31 between the asymptomatic (AS) and affected (AF) individuals of the AD5 family.
(A) PRPF31 mRNA expression normalized to the housekeeping gene GAPDH. Error bars refer to the standard deviation of the mean for 5 independent experiments for each group. (B) CNOT3 mRNA expression from the same 5 experiments used to generate PRPF31 data. **, p<0.01. (C) Linear regression analysis of PRPF31 and CNOT3 mRNA expression, which shows an inverse trend of the two genes in each cell line. Circles, asymptomatic subjects; triangles, affected individuals; open symbols, CNOT3 expression; Filled symbols, PRPF31 expression. Data having the same value for the x axis have been obtained from the same individual. (D) Quantification of CNOT3 protein abundance relative to β-actin from 3 independent SDS-PAGE gels, after simultaneous detection of the two proteins by quantitative LI-COR western blot.
Figure 2. CNOT3 silencing stimulates PRPF31 expression…
Figure 2. CNOT3 silencing stimulates PRPF31 expression in ARPE-19 cells.
(A) CNOT3 mRNA depletion by 2 different siRNA sequences and its effect on PRPF31 mRNA expression (B). ***, p<0.001. (C) Representative western blot of CNOT3 silencing and effect on PRPF31 protein expression. siRNA_1 and siRNA_2, different CNOT3-specific siRNA sequences; Control, treatment with transfection reagent with no siRNA; Mock, treatment with transfection reagent and scrambled siRNA.
Figure 3. CNOT3 binds to the PRPF31…
Figure 3. CNOT3 binds to the PRPF31 promoter in cells.
(A) CNOT3 ChIP-PCRs on different target sequences. Enrichment is visible only for PRPF31 promoter and CNOT3 promoter (positive control); DHFR 3′UTR, PTEN exon8, and GAPDH promoter sequences are all negative controls. (B) CNOT3 ChIP-q-PCR on PRPF31 promoter sequence. Error bars indicate the standard deviation of the mean for three independent ChIP-qPCR experiments. Serum IgG is used as IP negative control. ***, p<0.001.
Figure 4. Analysis of rs4806718 alleles in…
Figure 4. Analysis of rs4806718 alleles in two unrelated pedigrees.
(A) Family RP856/AD5. The individuals initially tested with NGS are marked with a star. The individuals marked with a triangle belong to a sibship pair, which was previously shown by McGee et al. to have the same isoallele haplotype but different phenotypes. (B) Family ADB1, a Bulgarian gypsy family carrying a heterozygous splice site mutation in PRPF31 (NM_015629.3:c.527+1G>T, or IVS6+1G>T). In both pedigrees carriers of mutations are either in black (affected individuals) or in grey (asymptomatic individuals).

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