In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors

Abstract

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

“…This approach utilizing minigenes harboring exons and the adjacent native introns (either full-length or abridged) has also been undertaken to assess defects of mRNA splicing caused by disease-associated variants in different genes, e . g ., PRPH2 [ 58 ], CLRN1 ( USH3A )[ 59 ], CNGβ1 [ 60 ]. A minimum of 200bp of intronic sequence from each end of the intron were used in generating abridged CFTR introns as this has been shown to be sufficient to capture the most critical splice regulatory sequences [ 61 , 62 ].…”

“…We have previously shown that the CFTR variant c.2988G>A, located in the last nucleotide of exon 18, results in partial missplicing [ 36 ] and a middle of exon ‘synonymous’ variant, c.2679G>T, has been reported to activate a cryptic splice site in CFTR exon 17 [ 68 ]. Importantly, these RNA-level effects of exonic variants have been reported in other disease-associated genes, such as ATP7B [ 69 ], DMD [ 15 ], and PRPH2 [ 58 ], thus our work emphasizes the need for careful evaluation of molecular consequences of exonic variants across the genome.…”

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

“…The majority of PRPH2 mutations are sequence variants. Different mechanisms, including aberrant mRNA splicing, protein mislocalization, and protein degradation may cause a reduced expression of the protein in the rod outer segment [72]. …”

Metabolic and redox signaling in the retina

“…RdCVFL is normally expressed by cones as well as rods in the mouse retina, while the trophic factor RdCVF is specifically expressed by rods: alternative splicing of the Nxnl1 gene is restricted to rods [ 127 ]. Differences between splicing pattern between rods and cones were revealed by the study of the PRPH2 gene [ 128 ]. The cones and their outer segments composed of polyunsaturated fatty acids are damaged by reactive oxygen species produced from leakage of the respiratory chain.…”