Recombinant Mouse U4/U6 small nuclear ribonucleoprotein Prp31 (Prpf31)

What is the primary function of Prpf31 in cellular processes?

Prpf31 serves as an essential pre-mRNA processing factor that mediates the assembly of the U4/U6.U5 tri-snRNP complex during pre-mRNA splicing. During each splicing cycle, Prpf31 facilitates the interaction between the U4/U6 di-snRNP and the U5 snRNP to form the functional tri-snRNP complex. Deletion of Prpf31 inhibits tri-snRNP assembly and consequently blocks pre-mRNA splicing, which is essential for cell viability . Beyond splicing, Prpf31 also participates in photoreceptor ciliogenesis, cellular adhesion, and supports critical retinal pigment epithelium (RPE) functions . When studying Prpf31 function, researchers should employ both splicing assays and cellular localization studies to comprehensively characterize its multifaceted roles.

How does haploinsufficiency manifest in Prpf31-related retinitis pigmentosa?

Prpf31-related retinitis pigmentosa (PRPF31-RP) exhibits an unusual "all or none" pattern of incomplete penetrance observed in most families carrying PRPF31 mutations. This phenomenon results from varying expression levels of the wild-type PRPF31 allele, which can differ by approximately 5-fold between individuals . Carriers of PRPF31 mutations may remain asymptomatic if their wild-type allele produces sufficient Prpf31 protein, but develop disease if Prpf31 levels fall below a critical threshold. This haploinsufficiency mechanism explains why some mutation carriers develop RP while others in the same family remain unaffected throughout their lives. Researchers investigating penetrance should quantify PRPF31 expression levels using qRT-PCR and protein quantification methods to establish correlations between expression levels and phenotypic manifestation.

Which genetic modifiers influence Prpf31 expression?

Several genetic elements modulate Prpf31 expression levels, contributing to the incomplete penetrance phenomenon. The MSR1 minisatellite repeat element, located upstream of the PRPF31 promoter, exists in either three or four copies, with the four-copy variant associated with non-penetrance . Another modifier gene, CNOT3, located downstream of PRPF31, also influences expression levels . When studying these modifiers, researchers should employ targeted sequencing of the promoter and surrounding regulatory regions, combined with expression analysis to determine how specific variants affect transcriptional activity. Chromatin immunoprecipitation (ChIP) assays can further elucidate how these genetic elements interact with transcription factors to modulate Prpf31 expression.

How do different mouse models of Prpf31 deficiency compare in phenotypic presentation?

Several mouse models have been developed to study PRPF31-RP, each with distinct characteristics as summarized in the following table:

Notably, conventional transgenic Prpf31+/- mice show primarily RPE defects rather than photoreceptor degeneration, which differs from the human disease . The AAV-CRISPR/Cas9 knockout models developed more recently demonstrate phenotypes more closely resembling human PRPF31-RP, particularly when delivered subretinally, making them potentially more suitable for therapeutic testing .

What are the advantages and limitations of using zebrafish for Prpf31 studies?

Zebrafish models provide unique advantages for studying Prpf31 function. Antisense morpholino knockdown of prpf31 in zebrafish has demonstrated that sublethal reduction of prpf31 results in defective photoreceptor outer segments and impaired visual function, evidenced by weakened optokinetic nystagmus .

Advantages:

• Transparent embryos allow direct observation of retinal development

• Rapid development enables faster experimental timelines

• Amenable to high-throughput drug screening

• Gene expression analysis revealed that many down-regulated genes were related to RP

Limitations:

• Complete prpf31 knockdown causes severe embryonic malformations and death

• Differences in retinal structure compared to mammals

• Transient knockdown using morpholinos may not fully recapitulate chronic disease states

• Some studies have shown conflicting results regarding which genes are mis-spliced

Researchers utilizing zebrafish should employ visual function assays such as optokinetic response measurements and electroretinography (ERG), combined with high-resolution imaging of retinal structure to comprehensively characterize phenotypes.

How can researchers establish non-human primate models for PRPF31-RP?

Establishing non-human primate (NHP) models represents an important step toward clinical translation of PRPF31-RP therapies. Recently, researchers have demonstrated the feasibility of creating NHP models using AAV-delivered CRISPR/Cas9 systems targeting PRPF31 . For efficient PRPF31 knockout in NHPs:

• Design species-specific gRNAs targeting early coding exons of PRPF31 (note that due to sequence differences, mouse gRNAs cannot be used for primates)

• Test gRNA efficiency in NHP retinal explants before in vivo application

• Package SaCas9 and validated gRNAs into AAV vectors with appropriate serotypes for retinal tropism

• Deliver vectors via subretinal injection to achieve targeted knockout in the outer retina

When evaluating NHP models, comprehensive assessment should include:

• Optical coherence tomography (OCT) to monitor structural changes

• Full-field and multifocal ERG to assess retinal function

• Fundus imaging to document retinal appearance

• Immunohistochemistry of retinal sections to evaluate cell-specific effects

Due to ethical and resource considerations, researchers should first validate their approaches in retinal explants before proceeding to live animal studies .

What methods are most effective for quantifying Prpf31 expression levels?

To accurately quantify Prpf31 expression levels, researchers should utilize a combination of techniques targeting both mRNA and protein:

For mRNA quantification:

• Quantitative RT-PCR with carefully designed primers spanning exon-exon junctions

• RNA-Seq for comprehensive transcriptome analysis, useful for detecting alternatively spliced variants

• Droplet digital PCR for absolute quantification with higher sensitivity

For protein quantification:

• Western blot with validated antibodies, using appropriate housekeeping proteins as controls

• Mass spectrometry-based proteomics for absolute quantification

• Immunofluorescence for assessing subcellular localization and expression patterns

When studying haploinsufficiency in PRPF31-RP, it's crucial to establish a correlation between expression levels and disease manifestation. Expression can vary by approximately 5-fold between individuals following a continuous distribution , so methodological consistency and multiple biological and technical replicates are essential for reliable quantification.

How can researchers assess the impact of Prpf31 mutations on splicing efficiency?

Evaluating the effects of Prpf31 mutations on splicing requires multiple complementary approaches:

• Mini-gene splicing assays: Design reporter constructs containing exons flanking an intron from genes known to be affected in PRPF31-RP (e.g., rhodopsin). Transfect these constructs along with wild-type or mutant Prpf31 into appropriate cell lines, then analyze splicing patterns using RT-PCR.

• RNA-Seq for global splicing analysis: Perform deep sequencing of the transcriptome from tissues or cells expressing wild-type or mutant Prpf31. Computational analysis should focus on:

  • Differential exon usage

  • Intron retention events

  • Alternative splice site selection

  • Analysis of specific retinal gene transcripts

• In vitro splicing assays: Prepare nuclear extracts from cells expressing wild-type or mutant Prpf31 and assess their ability to splice pre-mRNA substrates.

Previous studies have shown that transfection of PRPF31 mutants in mouse primary retinal cultures resulted in significantly decreased rhodopsin splicing, reduced rhodopsin expression, and rod apoptosis . Researchers should pay particular attention to genes involved in phototransduction, ciliogenesis, and retinal development when analyzing splicing defects.

What CRISPR/Cas9 strategies are most efficient for generating Prpf31 knockout models?

Based on recent research, the following CRISPR/Cas9 strategies have proven effective for generating Prpf31 knockout models:

For SpCas9-based approaches:

• Target early coding exons (particularly exons 2-3) of Prpf31

• Use gRNA2, which demonstrated 92.3 ± 0.58% editing efficiency in GFP-positive HEK293 cells

• Package SpCas9 and gRNA in separate AAV vectors due to size constraints

For SaCas9-based approaches (preferred due to packaging efficiency):

• Utilize a combination of gRNAs targeting multiple early exons for increased efficiency

• The combination of gRNAg (targeting exon 2) and gRNAh (targeting exon 3) demonstrated optimal efficiency, reducing Prpf31 expression to 39.00 ± 16.09% relative to controls in NIH/3T3 cells

• Package SaCas9 and dual gRNAs in a single AAV vector

Delivery considerations:

• Subretinal injection targets primarily outer retina (photoreceptors and RPE) and produces phenotypes more similar to human disease

• Intravitreal injection primarily affects inner retina

• Systemic delivery can be used for broader effects but may have off-target consequences

For validation of knockout efficiency, researchers should employ a combination of genomic DNA sequencing, mRNA expression analysis, protein quantification, and functional assessment of splicing activity .

Why do ubiquitously expressed splicing factors like Prpf31 cause retina-specific pathology?

The retina-specific pathology observed in PRPF31 mutations presents a fascinating paradox, as Prpf31 is ubiquitously expressed. Several hypotheses have emerged from research:

• Differential expression levels: Studies have shown that Prpf31 expression is higher in mouse RPE cells compared to neural retina cells , suggesting that RPE cells may have greater requirements for Prpf31 and thus increased sensitivity to reductions in Prpf31 levels.

• Photoreceptor-specific splicing demands: Photoreceptors have extraordinary metabolic and renewal demands, requiring continuous production of proteins involved in the visual cycle. This creates a heightened necessity for efficient splicing machinery.

• Retina-specific target genes: Immunoprecipitation and microarray studies have identified 146 genes that co-precipitate with Prpf31, with a considerable number being retina-specific . This suggests that Prpf31 may have specialized functions in processing retinal transcripts.

• Species-specific requirements: The mouse retina may not require as much Prpf31 as the human retina , which could explain differences in phenotypic manifestation between mouse models and human disease.

• RPE dysfunction as primary pathology: Evidence from multiple mouse models suggests that RPE dysfunction may precede photoreceptor degeneration , with defects in phagocytosis of shed photoreceptor outer segments potentially initiating the disease cascade.

Researchers investigating this question should consider comparative studies of Prpf31 function across different tissues, focusing on tissue-specific splicing patterns and requirements.

How do defects in RPE phagocytosis contribute to PRPF31-RP pathogenesis?

Defective phagocytosis in RPE cells has emerged as a potentially critical mechanism in PRPF31-RP pathogenesis. Studies of primary RPE cultures from Prpf31+/- mice have revealed:

• Disrupted diurnal rhythmicity of phagocytosis

• Mis-localization of phagocytic receptors

• Decreased phagocytic capacity specific to RPE cells (not observed in peritoneal macrophages)

The maintenance of normal retinal function relies on the regular elimination of shed photoreceptor outer segments by RPE cells . When this process is impaired:

• Undigested outer segment material accumulates

• RPE cells develop vacuolization and amorphous deposits between RPE and Bruch's membrane

• Basal infoldings of RPE cells are lost

• These RPE defects eventually lead to secondary photoreceptor degeneration

Similar phagocytic defects were observed when PRPF31 was knocked down in human RPE cells , supporting the relevance of this mechanism to human disease. Researchers investigating this pathway should employ phagocytosis assays with fluorescently labeled outer segments and time-course studies to characterize the dynamics of phagocytic dysfunction.

What cellular stress responses are activated in PRPF31-RP?

Multiple cellular stress pathways have been implicated in PRPF31-RP pathogenesis, providing potential therapeutic targets:

• Unfolded protein response (UPR): Studies of Prpf31+/A216P mice have shown upregulation of Hsp70 co-localized with mutant Prpf31 aggregates in RPE cytoplasm, indicating activation of the UPR . The unfolded protein response represents a cellular adaptation to endoplasmic reticulum stress caused by misfolded proteins.

• Oxidative stress responses: Differential expression analysis of RPE cells from Prpf31+/A216P mice identified upregulation of genes related to oxidative stress . This suggests that oxidative damage may contribute to RPE dysfunction in PRPF31-RP.

• DNA damage accumulation: Studies in zebrafish have shown that prpf31 deficiency results in abnormal expression of genes involved in DNA repair and homologous recombination, leading to the accumulation of DNA damage in retinal progenitor cells at early embryonic stages .

• Inflammatory pathways: Differential splicing analysis in Prpf31+/A216P mice demonstrated aberrant splicing of genes related to inflammation , suggesting that inflammatory processes may contribute to disease progression.

• Defective ciliogenesis: Prpf31 has been directly implicated in photoreceptor ciliogenesis . Genes involved in spindle formation, which also play roles in ciliogenesis, are affected by prpf31 deficiency, suggesting that defective cilia formation may contribute to photoreceptor degeneration.

Researchers investigating these stress responses should employ a combination of transcriptomic, proteomic, and functional assays to characterize pathway activation and identify potential intervention points.

What gene augmentation strategies show promise for treating PRPF31-RP?

AAV-mediated gene augmentation represents a promising approach for treating PRPF31-RP, given the haploinsufficiency disease mechanism. Recent research has demonstrated:

• Proof-of-concept in mouse models: Co-injection of AAV-Prpf31 with CRISPR/Cas9 knockout vectors in a rapidly degenerating mouse model restored retinal structure and function , providing the first in vivo evidence for the efficacy of gene augmentation therapy.

• Vector design considerations:

  • Promoter selection is critical; constitutive promoters like CAG or cell-specific promoters targeting photoreceptors and RPE should be evaluated

  • Vector capacity must accommodate the Prpf31 coding sequence (~1.5 kb)

  • AAV serotypes with tropism for photoreceptors and RPE (such as AAV2, AAV5, AAV8, or AAV9) are preferred

• Delivery parameters:

  • Subretinal injection appears most effective for targeting the relevant cell types

  • The therapeutic window remains to be fully defined; earlier intervention is likely more beneficial

  • Dose-response relationships need careful characterization to determine optimal viral titers

For researchers developing gene augmentation therapies, comprehensive outcome assessments should include:

• Structural analysis via OCT and histology

• Functional evaluation with ERG (a-, b-, and c-waves)

• Molecular confirmation of Prpf31 expression levels

• Assessment of splicing efficiency for retina-specific transcripts

How can genome editing approaches be applied to PRPF31-RP?

While gene augmentation addresses haploinsufficiency by supplying additional functional Prpf31, genome editing approaches offer alternative strategies:

• Allele-specific knockout of dominant negative mutations:

  • Design gRNAs that specifically target the mutant allele while sparing the wild-type allele

  • This approach is most applicable for mutations that exert dominant negative effects rather than simple haploinsufficiency

  • The specificity of allele discrimination is critical to avoid further reducing functional Prpf31 levels

• Upregulation of the wild-type allele:

  • CRISPR activation (CRISPRa) systems can target the endogenous wild-type Prpf31 promoter to enhance expression

  • This approach addresses the core haploinsufficiency mechanism by boosting output from the functional allele

  • Targeting modifier elements like the MSR1 minisatellite could potentially influence wild-type allele expression

• Correction of specific mutations:

  • Prime editing or base editing could correct point mutations without requiring double-strand breaks

  • Homology-directed repair using AAV-delivered templates could correct larger mutations

  • These approaches require highly efficient delivery to photoreceptors and RPE cells

Challenges specific to genome editing for PRPF31-RP include:

• Ensuring sufficient editing efficiency in the relevant cell types

• Minimizing off-target effects, particularly given the essential nature of splicing factors

• Packaging constraints of delivery vectors for the necessary editing machinery

What late-stage therapeutic interventions are available for advanced PRPF31-RP?

For patients with advanced PRPF31-RP where significant photoreceptor degeneration has already occurred, several late-stage interventions may provide benefit:

• Optogenetics:

  • Introduction of light-sensitive proteins into remaining retinal cells

  • Can potentially restore light sensitivity after photoreceptor loss

  • Requires specialized hardware (goggles) to amplify light signals

  • Clinical trials are ongoing for various forms of RP, with potential application to PRPF31-RP

• Cell transplantation:

  • Transplantation of photoreceptor precursors or retinal sheets

  • Derivation of photoreceptors from patient-specific iPSCs with genetic correction

  • Combined gene-cell therapy approaches where cells are genetically modified ex vivo

  • Faces challenges of cell integration and survival

• Retinal prostheses:

  • Electronic devices that stimulate remaining retinal neurons

  • Several approved devices available with different approaches to stimulation

  • Limited resolution but can restore basic visual functions

  • May be combined with gene therapy for enhanced outcomes

• Neuroprotective approaches:

  • Delivery of neurotrophic factors to slow photoreceptor degeneration

  • Anti-apoptotic strategies targeting cell death pathways

  • Reduction of oxidative stress or inflammation

  • These approaches aim to preserve remaining vision rather than restore lost function

For any late-stage intervention, patient selection and outcome expectations must be carefully managed. Assessment of retinal structure via OCT to identify remaining cells is essential for determining the most appropriate therapeutic approach.