Temporal and Tissue Specific Regulation of RP-Associated Splicing Factor Genes PRPF3, PRPF31 and PRPC8—Implications in the Pathogenesis of RP

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.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.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

Creation and characterization of mouse models of RNA splicing factor retinitis pigmentosa

The disorder retinitis pigmentosa (RP) is the most common inherited form of blindness. An interesting and currently unexplained form of RP is caused by mutations in ubiquitous RNA splicing factors essential for splicing of intron-containing pre-mRNA into mature mRNA. RNA splicing is an essential step in gene expression for most eukaryotic transcripts, yet despite the ubiquity of the mutated splicing factors causative in RP, only photoreceptors are affected. In order to study this form of RP we have created and characterized a number of animal models with mutations in the RNA splicing factors implicated in RP. We have created Prpf3 knockout mice, Prpf3-T494M knockin mice bearing the missense mutation found in RP18 patients, and Prpf8-H2309P knockin mice with a missense mutation found in RP13 in order to determine the role of the mutations in disease pathogenesis. We studied aspects of retinal structure and function, as well as the consequences of the mutant spliceosomal components on RNA splicing. We find that total knockout of these proteins is lethal, but heterozygous knockout produces no phenotype even at aged timepoints. In contrast, as the knockin animals age, the retina is less responsive to light, and the appearance of sub retinal deposits becomes prominent, indicating an unhealthy retina or retinal pigment epithelium. We also show for the first time in vivo that the knockin animals display alterations in RNA splicing of many transcripts in the retina, which demonstrates that the splicing factor mutations really do have consequences for RNA splicing. Furthermore we show that the alterations in RNA splicing are tissue-specific. Finally we observed that the steady state levels of snRNA are also altered in a tissue-specific way, which raises the possibility that the mutant splicing factors are altering the stability of the snRNP complexes, producing tissue specific effects on RNA splicing. We conclude that the mutations in Prpf3 and Prpf8 lead to RP through altering one or more vital transcripts in the retina or RPE, and propose studies to further characterize the retinal effects of these mutations and to identify the deleterious splicing alterations which are causative in these forms of RP

Creation and characterization of mouse models of RNA splicing factor retinitis pigmentosa

The disorder retinitis pigmentosa (RP) is the most common inherited form of blindness. An interesting and currently unexplained form of RP is caused by mutations in ubiquitous RNA splicing factors essential for splicing of intron-containing pre-mRNA into mature mRNA. RNA splicing is an essential step in gene expression for most eukaryotic transcripts, yet despite the ubiquity of the mutated splicing factors causative in RP, only photoreceptors are affected. In order to study this form of RP we have created and characterized a number of animal models with mutations in the RNA splicing factors implicated in RP. We have created Prpf3 knockout mice, Prpf3-T494M knockin mice bearing the missense mutation found in RP18 patients, and Prpf8-H2309P knockin mice with a missense mutation found in RP13 in order to determine the role of the mutations in disease pathogenesis. We studied aspects of retinal structure and function, as well as the consequences of the mutant spliceosomal components on RNA splicing. We find that total knockout of these proteins is lethal, but heterozygous knockout produces no phenotype even at aged timepoints. In contrast, as the knockin animals age, the retina is less responsive to light, and the appearance of sub retinal deposits becomes prominent, indicating an unhealthy retina or retinal pigment epithelium. We also show for the first time in vivo that the knockin animals display alterations in RNA splicing of many transcripts in the retina, which demonstrates that the splicing factor mutations really do have consequences for RNA splicing. Furthermore we show that the alterations in RNA splicing are tissue-specific. Finally we observed that the steady state levels of snRNA are also altered in a tissue-specific way, which raises the possibility that the mutant splicing factors are altering the stability of the snRNP complexes, producing tissue specific effects on RNA splicing. We conclude that the mutations in Prpf3 and Prpf8 lead to RP through altering one or more vital transcripts in the retina or RPE, and propose studies to further characterize the retinal effects of these mutations and to identify the deleterious splicing alterations which are causative in these forms of RP

Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration

To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice, and evaluated the retinal ultrastructure of Prpf31-knockout mice. All three mouse models demonstrate degenerative changes in the RPE with age, suggesting that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP