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S. S. Bhattacharya - One of the best experts on this subject based on the ideXlab platform.
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gene of the month PRPF31
Journal of Clinical Pathology, 2017Co-Authors: Anna M. Rose, Rong Luo, Utsav K Radia, S. S. BhattacharyaAbstract:Pre-mRNA splicing is an essential process in eukaryotic cells where the transcribed intronic sequences are removed, prior to translation into protein. PRPF31 is a ubiquitously expressed splicing factor, which aids in the assembly of the macromolecular spliceosome. Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa (adRP), a form of retinal degeneration that causes progressive visual impairment. Interestingly, mutations in PRPF31 are non-penetrant, with some mutation carriers being phenotypically unaffected. In this review, the gene organisation, protein structure and biological function of PRPF31 are discussed, and the mechanisms of non-penetrance in PRPF31-associated adRP are discussed.
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span poly l arginine nanoparticles are efficient non viral vectors for PRPF31 gene delivery an approach of gene therapy to treat retinitis pigmentosa
Nanomedicine: Nanotechnology Biology and Medicine, 2016Co-Authors: Andrea Pensado, Lourdes Valdessanchez, Berta De La Cerda, Ana B Garciadelgado, Francisco J Diazcorrales, Ana Aramburu Del Boz, Daniel Rodriguezmartinez, Begona Seijo, S. S. BhattacharyaAbstract:Retinitis pigmentosa (RP) is the most common cause of inherited blindness in adults. Mutations in the PRPF31 gene produce autosomal dominant RP (adRP). To date there are no effective treatments for this disease. The purpose of this study was to design an efficient non-viral vector for human PRPF31 gene delivery as an approach to treat this form of adRP. Span based nanoparticles were developed to mediate gene transfer in the subretinal space of a mouse model of adRP carrying a point mutation (A216P) in the PRPF31 gene. Funduscopic examination, electroretinogram, optomotor test and optical coherence tomography were conducted to further in vivo evaluate the safety and efficacy of the nanosystems developed. Span-polyarginine (SP-PA) nanoparticles were able to efficiently transfect the GFP and PRPF31 plasmid in mice retinas. Statistically significant improvement in visual acuity and retinal thickness were found in PRPF31A216P/+ mice treated with the SP-PA-PRPF31 nanomedicine.
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variant haploinsufficiency and phenotypic non penetrance in PRPF31 associated retinitis pigmentosa
Clinical Genetics, 2016Co-Authors: Anna M. Rose, S. S. BhattacharyaAbstract:Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited disorders, characterized by death of the retinal photoreceptor cells, leading to progressive visual impairment. One form of RP is caused by mutations in the ubiquitously expressed splicing factor, PRPF31, this form being known as RP11. An intriguing feature of RP11 is the presence of non-penetrance, which has been observed in the majority of PRPF31 mutation-carrying families. In contrast to variable expressivity, which is highly pervasive, true non-penetrance is a very rare phenomenon in Mendelian disorders. In this article, the molecular mechanisms underlying phenotypic non-penetrance in RP11 are explored. It is an elegant example of how our understanding of monogenic disorders has evolved from studying only the disease gene, to considering a mutation on the genetic background of the individual - the logical evolution in this genomic era.
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dominant PRPF31 mutations are hypostatic to a recessive cnot3 polymorphism in retinitis pigmentosa a novel phenomenon of linked trans acting epistasis
Annals of Human Genetics, 2014Co-Authors: Anna M. Rose, Amna Z. Shah, Giulia Venturini, Carlo Rivolta, Geoffrey E Rose, S. S. BhattacharyaAbstract:Mutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease. It has been suggested that a major modulating factor lies in close proximity to the wildtype PRPF31 gene on Chromosome 19, implying that a cis-acting factor directly alters PRPF31 expression. Variable expression of CNOT3 is one determinant of PRPF31 expression. This study explored the relationship between CNOT3 (a trans-acting factor) and its paradoxical cis-acting nature in relation to RP11. Linkage analysis on Chromosome 19 was performed in mutation-carrying families, and the inheritance of the wildtype PRPF31 allele in symptomatic-asymptomatic sibships was assessed-confirming that differential inheritance of wildtype chromosome 19q13 determines the clinical phenotype (P < 2.6 × 10(-7) ). A theoretical model was constructed that explains the apparent conflict between the linkage data and the recent demonstration that a trans-acting factor (CNOT3) is a major nonpenetrance factor: we propose that this apparently cis-acting effect arises due to the intimate linkage of CNOT3 and PRPF31 on Chromosome 19q13-a novel mechanism that we have termed "linked trans-acting epistasis."
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expression of PRPF31 and tfpt regulation in health and retinal disease
Human Molecular Genetics, 2012Co-Authors: Anna M. Rose, Amna Z. Shah, Christina Chakarova, Giovanna Alfano, Naushin Waseem, Razek Georges Coussa, Radwan Ajlan, Robert K Koenekoop, S. S. BhattacharyaAbstract:PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic‐asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency— the first report of this disease mechanism in adRP.
Adriana Buskin - One of the best experts on this subject based on the ideXlab platform.
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progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells the mechanism and its reversal through activation of autophagy
bioRxiv, 2021Co-Authors: Maria Georgiou, Adriana Buskin, Chunbo Yang, Robert Atkinson, Kuanting Pan, Marina Moya Molina, Joseph Collin, Jumana Y Alaama, Franziska Goertler, Sebastian LudwigAbstract:Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients. HighlightsO_LIPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival. C_LIO_LIMutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing. C_LIO_LIHigh-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells. C_LIO_LIAccumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells. C_LIO_LIProteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells. C_LIO_LIPRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation. C_LIO_LIPhagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells. C_LIO_LIActivation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival. C_LI
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aav mediated gene augmentation therapy restores critical functions in mutant PRPF31 ipsc derived rpe cells
Molecular therapy. Methods & clinical development, 2019Co-Authors: Elizabeth M Brydon, Eric A Pierce, Revital Bronstein, Adriana Buskin, Majlinda Lako, Rosario FernandezgodinoAbstract:Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild-type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSCs) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and it partially restored structure and barrier function. These results suggest that AAV-based gene therapy targeting RPE cells holds therapeutic promise for patients with PRPF31-related RP.
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aav mediated gene augmentation therapy restores critical functions in mutant ipsc derived PRPF31 cells
bioRxiv, 2019Co-Authors: Elizabeth M Brydon, Eric A Pierce, Revital Bronstein, Adriana Buskin, Majlinda Lako, Rosario FernandezgodinoAbstract:Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSC) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and partially restored structure and barrier function. These results provide proof-of concept that AAV-based gene therapy can be used to treat patients with PRPF31-related RP.
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disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa
Nature Communications, 2018Co-Authors: Adriana Buskin, Revital Bronstein, Joseph Collin, Valeria Chichagova, Basudha Basu, Sina Mozaffarijovin, David Dolan, Alastair Droop, Sudeep Mehrotra, Michael H FarkasAbstract:Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as PRPF31+/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and PRPF31+/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
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human ipsc derived rpe and retinal organoids reveal impaired alternative splicing of genes involved in pre mrna splicing in PRPF31 autosomal dominant retinitis pigmentosa type 11
Social Science Research Network, 2018Co-Authors: Adriana Buskin, Valeria Chichagova, Basudha Basu, Sina Mozaffarijovin, David Dolan, Alastair Droop, Lili Zhu, Joseph Colli, Revital Bronstei, Sudeep MehrotrAbstract:Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and PRPF31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.
David M Hunt - One of the best experts on this subject based on the ideXlab platform.
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disease mechanism for retinitis pigmentosa rp11 caused by missense mutations in the splicing factor gene PRPF31
Molecular Vision, 2008Co-Authors: Susan E Wilkie, S. S. Bhattacharya, Christina Chakarova, Kinga M Bujakowska, Veronika Vaclavik, Martin J Warren, David M HuntAbstract:Purpose Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function.
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a study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa adrp
Biochimica et Biophysica Acta, 2006Co-Authors: Susan E Wilkie, S. S. Bhattacharya, Martin J Warren, Keith J Morris, David M HuntAbstract:In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin beta1 for translocation to the nucleus, with no requirement for importin alpha1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with tau approximately 6 s and a slow component with tau approximately 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients.
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expression of PRPF31 mrna in patients with autosomal dominant retinitis pigmentosa a molecular clue for incomplete penetrance
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Eranga N Vithana, Leen Abusafieh, L Pelosini, Elizabeth Winchester, Dan Hornan, Alan C Bird, David M Hunt, Stephen A Bustin, S. S. BhattacharyaAbstract:PURPOSE. To investigate whether the incomplete penetrance phenotype characteristic of adRP families linked to chromosome 19q13.4 (RP11) with mutations in the PRPF31 gene is due to differentially expressed wild-type alleles in symptomatic and asymptomatic individuals. METHODS. Real-time quantitative RT-PCR was performed on RNA from lymphoblastoid cell lines derived from a large adRP family (RP856/AD5) that segregates an 11bp deletion in exon 11 of PRPF31. The mRNA levels from only the wild-type allele of PRPF31 were assayed using a probe designed across the deletion. The Mann–Whitney U test was used to compare the median mRNA copy numbers of the symptomatic with the asymptomatic carriers of the mutant PRPF31 allele. The PRPF31 protein levels from symptomatic and asymptomatic individuals were also assayed by Western blot analysis using an antibody specific to the wild-type PRPF31 protein. RESULTS. The use of cell lines was validated by the observation that cell transformation did not alter PRPF31 expression in the cell lines compared with nucleated blood cells and donor retinas. A significant difference in wild-type PRPF31 mRNA levels was observed between symptomatic and asymptomatic individuals (P 0.001) and was supported by Western blot analysis of the PRPF31 protein. CONCLUSIONS. Partial penetrance in RP11 could be due to the coinheritance of a PRPF31 gene defect and a low-expressed wild-type allele. This study revealed a potential avenue for future therapy in that it appears the moderate overexpression of wild-type PRPF31 may prevent clinical manifestation of the disease. (Invest Ophthalmol Vis Sci. 2003;44:4204 – 4209) DOI:10.1167/iovs.03-0253 R etinitis pigmentosa is a clinically and genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and visual field loss. Clinical manifestations include pigment deposition in the retina and attenuation of retinal blood vessels, with later depigmentation or atrophy of the RPE. ERG abnormalities are recordable in the early stages with attenuation of rod and cone responses. In advanced RP both rod and cone ERG responses are extinguished. 1 With an incidence of approximately 1 in 3500, RP can be inherited as an X-linked, autosomal dominant or autosomal recessive condition. 1
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disease mechanism for retinitis pigmentosa rp11 caused by mutations in the splicing factor gene PRPF31
Human Molecular Genetics, 2002Co-Authors: Evelyne Deery, S. S. Bhattacharya, Eranga N Vithana, David M Hunt, Martin J Warren, Richard J Newbold, Victoria A Gallon, Susan E WilkieAbstract:This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology.
Eric A Pierce - One of the best experts on this subject based on the ideXlab platform.
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Decreased Levels of the RNA Splicing Factor Prpf3 in Mice and Zebrafish Do Not Cause Photoreceptor Degeneration
2020Co-Authors: John J Graziotto, Eric A Pierce, Chris F Inglehearn, Michael A Pack, F M KirbyAbstract:PURPOSE. Pre-mRNA processing factor 3 (PRPF3) is a spliceosomal component essential for pre-mRNA processing. Mutations in PRPF3 have been implicated in retinitis pigmentosa (RP) 18 through an unknown mechanism. The authors created and characterized Prpf3 knockout mice and zebrafish to determine whether RP18 is a result of haploinsufficiency. METHODS. Mice were produced from a Prpf3 gene trap cell line, and parameters of retinal function, structure, and RNA splicing were analyzed. The retinas of prpf3 insertional mutant zebrafish were also analyzed histologically. RESULTS. Homozygous Prpf3 knockout mice do not survive to 14 days postfertilization (dpf), implying that this allele is required for early embryonic development. Homozygous Prpf3 knockout zebrafish die by 4dpf, well beyond the mid-blastula transition at which transcription activates. Zebrafish knockout embryos reveal abnormally high levels of cell death in the developing eye. Heterozygous Prpf3 knockout mice have less than the expected 50% reduction in Prpf3 at the mRNA and protein levels, implying compensatory expression from the wild-type allele. The heterozygous mice develop normally, with no changes in retinal function, no evidence for photoreceptor degeneration at up to 23 months of age, and no decrease in pre-mRNA splicing of transcripts mutated in other forms of RP in the retina. Similarly, heterozygous prpf3 knockout zebrafish develop normally and show no retinal degeneration up to 12 months of age. CONCLUSIONS. These models suggest that RP18 is not a result of haploinsufficiency but instead arises from a toxic gain of function caused by missense mutations in PRPF3. (Invest Ophthalmol Vis Sci. 2008;49:3830 -3838) DOI:10.1167/iovs.07-1483 A ubiquitous process in eukaryotic cells, pre-mRNA splicing is an essential step in gene expression. It takes place in a large ribonucleoprotein complex called the spliceosome, which, in addition to the pre-mRNA substrate, is composed of five small nuclear ribonucleoprotein complexes (snRNPs), U1, U2, U4/U6, and U5, and a host of non-snRNP accessory proteins (for reviews, see Krämer 1 and Krainer 2 ). Pre-mRNA processing factor 3 (PRPF3) protein is associated with the U4/U6 snRNP complex and is necessary for the integrity of the U4/U6/U5 tri-snRNP complex, without which splicing cannot occur. 6 -9 The C terminus is the most highly conserved region of PRPF3, suggesting it has an important function. 3 In recent years, two missense mutations in the highly conserved C terminus of PRPF3 and mutations in three other spliceosomal proteins have been implicated in autosomal dominant retinitis pigmentosa (adRP). 15,16 RP patients experience progressive night blindness because of the loss of rod photoreceptor cells of the retina, followed by loss of peripheral vision and eventual blindness resulting from secondary degeneration of cones later in life. 17 RP is genetically heterogeneous and can be inherited by autosomal dominant (adRP), autosomal recessive (arRP), or X-linked (xlRP) transmission. 16 Despite this heterogeneity, most of the genes implicated in RP are expressed specifically in photoreceptor cells and encode proteins involved in the phototransduction cascade, photoreceptor structure, or other components of known visual pathways. 18 The discovery that 4 of the 14 known forms of dominant RP are caused by mutations in splicing factors suggests a novel and unexpected pathway to retinal degeneration. However, it is unclear how mutations in these ubiquitously expressed splicing factors lead to retina-specific disease. This question is of particular importance because, as a group, the RNA splicing factor forms of RP are the second most common cause of RP; the first is RP caused by mutations in rhodopsin
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aav mediated gene augmentation therapy restores critical functions in mutant PRPF31 ipsc derived rpe cells
Molecular therapy. Methods & clinical development, 2019Co-Authors: Elizabeth M Brydon, Eric A Pierce, Revital Bronstein, Adriana Buskin, Majlinda Lako, Rosario FernandezgodinoAbstract:Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild-type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSCs) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and it partially restored structure and barrier function. These results suggest that AAV-based gene therapy targeting RPE cells holds therapeutic promise for patients with PRPF31-related RP.
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aav mediated gene augmentation therapy restores critical functions in mutant ipsc derived PRPF31 cells
bioRxiv, 2019Co-Authors: Elizabeth M Brydon, Eric A Pierce, Revital Bronstein, Adriana Buskin, Majlinda Lako, Rosario FernandezgodinoAbstract:Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSC) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and partially restored structure and barrier function. These results provide proof-of concept that AAV-based gene therapy can be used to treat patients with PRPF31-related RP.
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Course of Ocular Function in PRPF31 Retinitis Pigmentosa.
Seminars in Ophthalmology, 2016Co-Authors: Brian Hafler, Jason Comander, Carol Weigel Difranco, Emily Place, Eric A PierceAbstract:ABSTRACTMutations in pre-mRNA splicing factors are the second most common cause of autosomal dominant retinitis pigmentosa, and a major cause of vision loss. The development of gene augmentation therapy for disease caused by mutations in PRPF31 necessitates defining pretreatment characteristics and disease progression of patients with PRPF31-related retinitis pigmentosa. We show rates of decline of visual field area −6.9% per year and 30-Hz flicker cone response of −9.2% per year, which are both similar to observed rates for retinitis pigmentosa. We hypothesize that RNA splicing factor retinitis pigmentosa will be amenable to treatment by AAV-mediated gene therapy, and that understanding the clinical progression rates of PRPF31 retinitis pigmentosa will help with the design of gene therapy clinical trials.
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temporal and tissue specific regulation of rp associated splicing factor genes prpf3 PRPF31 and prpc8 implications in the pathogenesis of rp
PLOS ONE, 2011Co-Authors: Huibi Cao, Robert S Molday, Simon Lam, Rongqi Duan, Catherine Newnham, John J Graziotto, Eric A PierceAbstract:Background Genetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors. Methodology/Principal Findings We examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells. Conclusions/Significance Our data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein.
Susan E Wilkie - One of the best experts on this subject based on the ideXlab platform.
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disease mechanism for retinitis pigmentosa rp11 caused by missense mutations in the splicing factor gene PRPF31
Molecular Vision, 2008Co-Authors: Susan E Wilkie, S. S. Bhattacharya, Christina Chakarova, Kinga M Bujakowska, Veronika Vaclavik, Martin J Warren, David M HuntAbstract:Purpose Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function.
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a study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa adrp
Biochimica et Biophysica Acta, 2006Co-Authors: Susan E Wilkie, S. S. Bhattacharya, Martin J Warren, Keith J Morris, David M HuntAbstract:In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin beta1 for translocation to the nucleus, with no requirement for importin alpha1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with tau approximately 6 s and a slow component with tau approximately 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients.
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disease mechanism for retinitis pigmentosa rp11 caused by mutations in the splicing factor gene PRPF31
Human Molecular Genetics, 2002Co-Authors: Evelyne Deery, S. S. Bhattacharya, Eranga N Vithana, David M Hunt, Martin J Warren, Richard J Newbold, Victoria A Gallon, Susan E WilkieAbstract:This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology.
