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.
References
[1]
Goldberg AF. Role of peripherin/rds in vertebrate photoreceptor architecture and inherited retinal degenerations. Int Rev Cytol. 2006;253: 131–175. pmid:17098056 doi: 10.1016/s0074-7696(06)53004-9
[2]
Conley SM, Stuck MW, Naash MI. Structural and functional relationships between photoreceptor tetraspanins and other superfamily members. Cell Mol Life Sci. 2012;69(7): 1035–1047. doi: 10.1007/s00018-011-0736-0. pmid:21655915
[3]
Boon CJ, den Hollander AI, Hoyng CB, Cremers FP, Klevering BJ, Keunen JE. The spectrum of retinal dystrophies caused by mutations in the peripherin/RDS gene. Prog Retin Eye Res. 2008;27(2): 213–235. doi: 10.1016/j.preteyeres.2008.01.002. pmid:18328765
[4]
Wang GS, Cooper TA. Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet. 2007;8(10): 749–761. pmid:17726481 doi: 10.1038/nrg2164
[5]
Becirovic E, Ebermann I, Nagy D, Zrenner E, Seeliger MW, Bolz HJ. Usher syndrome type 1 due to missense mutations on both CDH23 alleles: investigation of mRNA splicing. Hum Mutat. 2008;29(3): 452. doi: 10.1002/humu.9526
[6]
Cartegni L, Krainer AR. Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet. 2002;30(4): 377–384. pmid:11925564 doi: 10.1038/ng854
[7]
Liu HX, Cartegni L, Zhang MQ, Krainer AR. A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet. 2001;27(1): 55–58. pmid:11137998
[8]
Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. 2002;3(4): 285–298. pmid:11967553 doi: 10.1038/nrg775
[9]
Fairbrother WG, Yeh RF, Sharp PA, Burge CB. Predictive identification of exonic splicing enhancers in human genes. Science. 2002;297(5583): 1007–1013. pmid:12114529 doi: 10.1126/science.1073774
[10]
Pagani F, Baralle FE. Genomic variants in exons and introns: identifying the splicing spoilers. Nat Rev Genet. 2004;5(5): 389–396. pmid:15168696 doi: 10.1038/nrg1327
[11]
Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, et al. Alternative isoform regulation in human tissue transcriptomes. Nature. 2008;456(7221): 470–476. doi: 10.1038/nature07509. pmid:18978772
[12]
Lewandowska MA. The missing puzzle piece: splicing mutations. Int J Clin Exp Pathol. 2013;6(12): 2675–2682. pmid:24294354
[13]
Trapani I, Puppo A, Auricchio A. Vector platforms for gene therapy of inherited retinopathies. Prog Retin Eye Res. 2014;43C: 108–128. doi: 10.1016/j.preteyeres.2014.08.001
[14]
Petrs-Silva H, Dinculescu A, Li Q, Min SH, Chiodo V, Pang JJ, et al. High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Ther. 2009;17(3): 463–471. doi: 10.1038/mt.2008.269. pmid:19066593
[15]
Yanagihashi S, Nakazawa M, Kurotaki J, Sato M, Miyagawa Y, Ohguro H. Autosomal dominant central areolar choroidal dystrophy and a novel Arg195Leu mutation in the peripherin/RDS gene. Arch Ophthalmol. 2003;121(10): 1458–1461. pmid:14557183 doi: 10.1001/archopht.121.10.1458
[16]
Sullivan LS, Bowne SJ, Birch DG, Hughbanks-Wheaton D, Heckenlively JR, Lewis RA, et al. Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. Invest Ophthalmol Vis Sci. 2006;47(7): 3052–3064. pmid:16799052 doi: 10.1167/iovs.05-1443
[17]
Coco RM, Telleria JJ, Sanabria MR, Rodriguez-Rua E, Garcia MT. PRPH2 (Peripherin/RDS) mutations associated with different macular dystrophies in a Spanish population: a new mutation. Eur J Ophthalmol. 2010;20(4): 724–732. pmid:20213611
[18]
Budu , Hayasaka S, Matsumoto M, Yamada T, Zhang XY, Hayasaka Y. Peripherin/RDS gene mutation (Pro210Leu) and polymorphisms in Japanese patients with retinal dystrophies. Jpn J Ophthalmol. 2001;45(4): 355–358. pmid:11485765 doi: 10.1016/s0021-5155(01)00334-3
[19]
Felbor U, Schilling H, Weber BH. Adult vitelliform macular dystrophy is frequently associated with mutations in the peripherin/RDS gene. Hum Mutat. 1997;10(4): 301–309. pmid:9338584 doi: 10.1002/(sici)1098-1004(1997)10:4<301::aid-humu6>3.3.co;2-y
[20]
Saga M, Mashima Y, Akeo K, Oguchi Y, Kudoh J, Shimizu N. A novel Cys-214-Ser mutation in the peripherin/RDS gene in a Japanese family with autosomal dominant retinitis pigmentosa. Hum Genet. 1993;92(5): 519–521. pmid:8244346 doi: 10.1007/bf00216463
[21]
Payne AM, Downes SM, Bessant DA, Bird AC, Bhattacharya SS. Founder effect, seen in the British population, of the 172 peripherin/RDS mutation-and further refinement of genetic positioning of the peripherin/RDS gene. Am J Hum Genet. 1998;62(1): 192–195. pmid:9443872 doi: 10.1086/301679
[22]
Jacobson SG, Cideciyan AV, Kemp CM, Sheffield VC, Stone EM. Photoreceptor function in heterozygotes with insertion or deletion mutations in the RDS gene. Invest Ophthalmol Vis Sci. 1996;37(8): 1662–1674. pmid:8675410
[23]
Rodriguez JA, Gannon AM, Daiger SP. Screening for mutations in rhodopsin and peripherin/RDS in patients with autosomal dominant retinitis pigmentosa. Am J Hum Genet. 1994;55(Suppl.3): 44. .
[24]
Kohl S, Christ-Adler M, Apfelstedt-Sylla E, Kellner U, Eckstein A, Zrenner E, et al. RDS/peripherin gene mutations are frequent causes of central retinal dystrophies. J Med Genet. 1997;34(8): 620–626. pmid:9279751 doi: 10.1136/jmg.34.8.620
[25]
Renner AB, Fiebig BS, Weber BH, Wissinger B, Andreasson S, Gal A, et al. Phenotypic variability and long-term follow-up of patients with known and novel PRPH2/RDS gene mutations. Am J Ophthalmol. 2009;147(3): 518–530 e511. doi: 10.1016/j.ajo.2008.09.007. pmid:19038374
[26]
Akimoto M, Cheng H, Zhu D, Brzezinski JA, Khanna R, Filippova E, et al. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Proc Natl Acad Sci U S A. 2006;103(10): 3890–3895. pmid:16505381 doi: 10.1073/pnas.0508214103
[27]
Fei Y, Hughes TE. Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse. Vis Neurosci. 2001;18(4): 615–623. pmid:11829307 doi: 10.1017/s0952523801184117
[28]
Becirovic E, Nguyen ON, Paparizos C, Butz ES, Stern-Schneider G, Wolfrum U, et al. Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors. Hum Mol Genet. 2014;23(22): 5989–5997. doi: 10.1093/hmg/ddu323. pmid:24963162
[29]
Stricker HM, Ding XQ, Quiambao A, Fliesler SJ, Naash MI. The Cys214—>Ser mutation in peripherin/rds causes a loss-of-function phenotype in transgenic mice. Biochem J. 2005;388(Pt 2): 605–613. pmid:15656787 doi: 10.1042/bj20041960
[30]
Nour M, Ding XQ, Stricker H, Fliesler SJ, Naash MI. Modulating expression of peripherin/rds in transgenic mice: critical levels and the effect of overexpression. Invest Ophthalmol Vis Sci. 2004;45(8): 2514–2521. pmid:15277471 doi: 10.1167/iovs.04-0065
[31]
Ding XQ, Nour M, Ritter LM, Goldberg AF, Fliesler SJ, Naash MI. The R172W mutation in peripherin/rds causes a cone-rod dystrophy in transgenic mice. Hum Mol Genet. 2004;13(18): 2075–2087. pmid:15254014 doi: 10.1093/hmg/ddh211
[32]
Murphy D, Singh R, Kolandaivelu S, Ramamurthy V, Stoilov P. Alternative Splicing Shapes the Phenotype of a Mutation in BBS8 To Cause Nonsyndromic Retinitis Pigmentosa. Mol Cell Biol. 2015;35(10): 1860–1870. doi: 10.1128/MCB.00040-15. pmid:25776555
[33]
Siegert S, Cabuy E, Scherf BG, Kohler H, Panda S, Le YZ, et al. Transcriptional code and disease map for adult retinal cell types. Nat Neurosci. 2012;15(3): 487–495, S481-482. doi: 10.1038/nn.3032. pmid:22267162
[34]
Wang Y, Xiao X, Zhang J, Choudhury R, Robertson A, Li K, et al. A complex network of factors with overlapping affinities represses splicing through intronic elements. Nat Struct Mol Biol. 2013;20(1): 36–45. doi: 10.1038/nsmb.2459. pmid:23241926
[35]
Wang Y, Ma M, Xiao X, Wang Z. Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules. Nat Struct Mol Biol. 2012;19(10): 1044–1052. doi: 10.1038/nsmb.2377. pmid:22983564
[36]
Koch S, Sothilingam V, Garcia Garrido M, Tanimoto N, Becirovic E, Koch F, et al. Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa. Hum Mol Genet. 2012;21(20): 4486–4496. pmid:22802073 doi: 10.1093/hmg/dds290
[37]
Michalakis S, Mühlfriedel R, Tanimoto N, Krishnamoorthy V, Koch S, Fischer MD, et al. Restoration of cone vision in the CNGA3-/- mouse model of congenital complete lack of cone photoreceptor function. Mol Ther. 2010;18(12): 2057–2063. doi: 10.1038/mt.2010.149. pmid:20628362
[38]
Michalakis S, Geiger H, Haverkamp S, Hofmann F, Gerstner A, Biel M. Impaired opsin targeting and cone photoreceptor migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. Invest Ophthalmol Vis Sci. 2005;46(4): 1516–1524. pmid:15790924 doi: 10.1167/iovs.04-1503
[39]
Huttl S, Michalakis S, Seeliger M, Luo DG, Acar N, Geiger H, et al. Impaired channel targeting and retinal degeneration in mice lacking the cyclic nucleotide-gated channel subunit CNGB1. J Neurosci. 2005;25(1): 130–138. pmid:15634774 doi: 10.1523/jneurosci.3764-04.2005
[40]
Much B, Wahl-Schott C, Zong X, Schneider A, Baumann L, Moosmang S, et al. Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem. 2003;278(44): 43781–43786. pmid:12928435 doi: 10.1074/jbc.m306958200
