Recombinant Danio rerio U4/U6 small nuclear ribonucleoprotein Prp31 (prpf31), partial

What is the functional role of Prpf31 in zebrafish and how does it compare to human PRPF31?

Prpf31 in zebrafish (Danio rerio) functions as an essential component of the pre-mRNA splicing machinery, specifically within the U4/U6 small nuclear ribonucleoprotein complex. Zebrafish Prpf31 (DrPrpf31) contains 508 amino acids and shares remarkably high similarity (82.0%) with human PRPF31 . This conservation extends to functional domains including the Nop domain that mediates protein-RNA interactions .

Functionally, Prpf31 plays a critical role in tri-snRNP assembly by tethering U5 snRNP to the U4/U6 snRNP, facilitating spliceosome formation . Studies have demonstrated that this protein is essential for:

• Embryonic development (homozygous knockout is embryonically lethal)

• Retinal photoreceptor maintenance

• Proper splicing of retina-specific transcripts

• Hematopoietic stem and progenitor cell (HSPC) expansion

The high sequence and functional conservation between species makes zebrafish an excellent model for studying human PRPF31-related disorders, particularly autosomal dominant retinitis pigmentosa (adRP).

How can researchers effectively generate and validate Prpf31 knockout or mutant zebrafish models?

Researchers can employ several complementary approaches to generate and validate Prpf31 knockout or mutant zebrafish models:

Generation Methods:

• Morpholino oligonucleotides (MOs): Translation-blocking MOs against prpf31 can be injected into 1-cell stage embryos. This approach allows for dose-dependent knockdown, with low doses resulting in isolated visual defects (optokinetic nystagmus deficiency) and high doses causing broad malformations and embryonic lethality .

• CRISPR/Cas9 genome editing: Multiple studies have successfully generated prpf31 knockout lines using CRISPR/Cas9. For example:

  • SaCas9 with dual guide RNAs (gRNAg targeting exon 2 and gRNAh targeting exon 3) has achieved efficient knockout

  • Specific mutations can be introduced to mimic human disease variants

• Transgenic expression of mutant variants: For studying dominant-negative or haploinsufficiency effects, researchers can inject mRNAs encoding wild-type or mutant Prpf31 into zebrafish embryos .

Validation Methods:

• Western blot analysis: To confirm protein reduction using anti-PRPF31 antibodies

• qRT-PCR: To assess endogenous prpf31 expression levels

• Deep sequencing: To quantify genomic modification rates (e.g., 38.02 ± 12.66% of mPrpf31 modified in rescue studies)

• Phenotypic assessment: Evaluating embryonic development, mortality rates, and vision-related phenotypes

• Fundoscopy and OCT imaging: To monitor retinal degeneration in vivo

When designing CRISPR knockout strategies, researchers should note that complete homozygous knockout of prpf31 is embryonically lethal, so conditional or tissue-specific approaches may be necessary for studying adult functions.

What are the key differences between the SP117 and AD5 Prpf31 mutants in zebrafish models?

The SP117 and AD5 mutants represent two distinct PRPF31 mutations associated with human retinitis pigmentosa that exhibit fundamentally different pathogenic mechanisms when modeled in zebrafish:

These differences highlight how distinct mutations in the same gene can lead to retinal degeneration through different mechanisms, informing potential therapeutic approaches for PRPF31-associated retinal diseases.

How can researchers assess splicing defects in Prpf31 mutant zebrafish models?

Researchers can employ a multi-faceted approach to comprehensively assess splicing defects in Prpf31 mutant zebrafish models:

• RNA-Seq and transcriptome analysis:

  • Perform deep sequencing of retinal RNA to identify differentially expressed and alternatively spliced transcripts

  • Analyze intron retention events, which are particularly common in Prpf31 mutants

  • Compare splicing patterns between wild-type and mutant samples to identify affected transcripts

• RT-PCR validation of specific target genes:

  • Design primers flanking exon-intron boundaries of candidate genes

  • Focus on retina-specific transcripts known to be affected in Prpf31 models, such as:

    • Phototransduction genes (rho, gnat1, gnat2, pde6c)

    • Signal transduction genes (guk1, rcv1)

    • Calcium binding proteins (calb2)

• Quantitative assessment of retained introns:

  • Use qRT-PCR with intron-specific primers to quantify intron retention levels

  • Calculate splicing efficiency by comparing the ratio of spliced to unspliced transcripts

• In vivo splicing reporter assays:

  • Generate fluorescent reporter constructs containing introns from affected genes

  • Assess splicing efficiency in live zebrafish embryos or retinal cells

• Nuclear vs. cytoplasmic RNA fractionation:

  • Separate nuclear and cytoplasmic RNA fractions to detect nuclear accumulation of improperly spliced transcripts

  • This can reveal retention of intron-containing transcripts in the nucleus, a hallmark of splicing defects

• Immunofluorescence for splicing factors:

  • Examine the localization of spliceosomal components in photoreceptor cells

  • Determine if mutant Prpf31 affects the distribution of other splicing factors

• dsRNA accumulation analysis:

  • Staining with anti-dsRNA antibodies (such as J2) can detect double-stranded RNA accumulation resulting from splicing defects

  • This is particularly relevant as aberrant splicing can lead to dsRNA formation and potential triggering of innate immune responses

When interpreting results, researchers should consider that different mutations (like AD5 vs. SP117) may affect splicing patterns differently, with AD5 exhibiting more severe splicing defects consistent with its dominant-negative effect .

What are the most effective experimental approaches to study the relationship between Prpf31 dysfunction and retinal degeneration?

To effectively study the relationship between Prpf31 dysfunction and retinal degeneration, researchers should employ a comprehensive, multi-level experimental approach:

• In vivo retinal phenotyping:

  • Fundoscopy: Track development of retinal pallor, pigmentary changes, and vascular attenuation characteristic of RP

  • Optical Coherence Tomography (OCT): Quantify retinal layer thickness, particularly the outer nuclear layer (ONL), inner/outer segments (IS/OS), and RPE

  • Optokinetic nystagmus (OKN) response: Assess visual function in zebrafish larvae

  • Electroretinography (ERG): Measure electrical responses of photoreceptors and other retinal neurons

• Cellular and subcellular analysis:

  • Immunohistochemistry of retinal sections: Evaluate photoreceptor morphology and protein localization

  • TUNEL assay: Quantify apoptotic cells in the retina

  • Live imaging of fluorescently tagged Prpf31: Track protein localization and dynamics

• Molecular mechanism investigation:

  • RNA-seq analysis: Identify differentially expressed and alternatively spliced transcripts

  • Proteomics: Examine changes in protein expression and post-translational modifications

  • Co-immunoprecipitation: Study interactions between Prpf31 and other spliceosomal components

  • Protein stability assays: Compare degradation rates of wild-type and mutant proteins

• Temporally-controlled gene manipulation:

  • Inducible CRISPR systems: Allow for timed knockout to distinguish developmental from maintenance roles

  • Heat-shock promoters: Control transgene expression at specific developmental stages

• Cell-type specific approaches:

  • Conditional knockouts in photoreceptors vs. RPE: Determine primary site of pathology

  • Single-cell RNA-seq: Identify cell-type specific responses to Prpf31 dysfunction

• Therapeutic testing platforms:

  • AAV-mediated gene augmentation: Assess rescue of retinal structure and function

  • Co-injection rescue experiments: Test if wild-type Prpf31 can overcome mutant phenotypes

• Cross-species validation:

  • Comparison with mouse models: Validate findings across vertebrate systems

  • iPSC-derived retinal organoids from patients: Translate findings to human cells

This integrated approach has revealed key insights, such as the distinct pathomechanisms of different PRPF31 mutations (haploinsufficiency for SP117 vs. dominant-negative effects for AD5) , and the tissue-specific sensitivity to reduced Prpf31 levels despite its ubiquitous expression .

How does Prpf31 dysfunction specifically affect retinal photoreceptors despite being a ubiquitously expressed splicing factor?

The selective vulnerability of retinal photoreceptors to Prpf31 dysfunction despite its ubiquitous expression represents a central paradox in PRPF31-associated retinitis pigmentosa. Research has revealed several mechanisms that explain this tissue-specific pathology:

• Heightened splicing demands in photoreceptors:

  • Photoreceptors have extraordinarily high metabolic and biosynthetic activity, creating elevated demands for efficient pre-mRNA splicing

  • The daily renewal of approximately 10% of outer segments requires intensive transcription and processing of photoreceptor-specific genes

  • This creates a "splicing stress" scenario where reduced PRPF31 function becomes limiting in photoreceptors before other cell types

• Selective impact on retina-specific transcripts:

  • Gene expression analysis in zebrafish prpf31 morphants reveals selective downregulation of retina-specific genes including:

    • Rhodopsin (rho)

    • G protein subunits (gnat1, gnat2)

    • Phosphodiesterase (pde6c)

    • Other phototransduction components (guk1, rcv1, calb2)

  • These transcripts may be particularly dependent on optimal Prpf31 function for proper splicing

• Threshold effect in haploinsufficiency:

  • In PRPF31 mutation carriers, the remaining PRPF31 from the wild-type allele appears sufficient for normal function in most tissues

  • Photoreceptors may require higher baseline levels of PRPF31, making them uniquely sensitive to reduced dosage

  • Evidence from zebrafish studies shows that SP117 mutation leads to haploinsufficiency without photoreceptor degeneration upon overexpression, suggesting that a critical threshold of functional protein must be maintained

• Differential sensitivity to dominant-negative effects:

  • The AD5 mutation demonstrates a dominant-negative mechanism in zebrafish models

  • This mutation may selectively interfere with splicing complexes in photoreceptors due to their unique splicing requirements

• RPE involvement amplifying photoreceptor damage:

  • Studies in mouse models suggest that PRPF31 mutations may primarily affect the retinal pigment epithelium (RPE)

  • RPE dysfunction can secondarily lead to photoreceptor degeneration due to their interdependent relationship

• Specialized splicing regulation in photoreceptors:

  • Photoreceptors may utilize specialized splicing regulatory mechanisms that are particularly dependent on optimal Prpf31 function

  • This could explain why certain mutations have selective effects on photoreceptor-specific transcripts

These findings highlight the concept of "relative insufficiency," where ubiquitously expressed splicing factors like Prpf31 become limiting specifically in tissues with extraordinary splicing demands, providing a model for understanding why mutations in ubiquitous splicing factors can lead to retina-specific pathology.

What methods can be used to differentiate between haploinsufficiency and dominant-negative effects of Prpf31 mutations?

Differentiating between haploinsufficiency and dominant-negative effects of Prpf31 mutations requires a systematic experimental approach combining genetic, molecular, and cellular techniques:

• Overexpression studies in wild-type backgrounds:

  • Principle: Dominant-negative mutations will cause phenotypes when overexpressed in wild-type backgrounds, while haploinsufficient mutations typically will not

  • Evidence: In zebrafish, AD5 mRNA injection into wild-type embryos caused dose-dependent malformations and lethality, while SP117 mRNA injection had no adverse effects, supporting dominant-negative and haploinsufficiency mechanisms, respectively

• Co-injection with morpholinos:

  • Principle: Dominant-negative mutations will exacerbate morpholino knockdown phenotypes

  • Method: Inject prpf31 morpholinos together with mutant mRNAs and assess phenotypic severity

  • Results: Co-injection of AD5 mRNA with prpf31 MO significantly increased embryonic lethality (62.2% vs. 22.7% with AD5 alone), providing evidence for dominant-negative activity

• Rescue experiments:

  • Approach: Test whether wild-type Prpf31 can rescue phenotypes caused by mutant Prpf31

  • Findings: Wild-type prpf31 rescued embryonic lethality induced by AD5, consistent with dominant-negative effects that can be overcome by increasing wild-type protein levels

  • Quantification: Measure rescue efficiency across different wild-type:mutant ratios

• Protein localization studies:

  • Technique: Fluorescently tag wild-type and mutant Prpf31 and track subcellular localization

  • Observations: SP117 protein showed aberrant cytoplasmic localization in rod photoreceptors, while AD5 was initially nuclear but later found in the cytoplasm concurrent with rod degeneration

• Protein-protein interaction analysis:

  • Methods: Co-immunoprecipitation, proximity ligation assays, or FRET

  • Target: Assess if mutant Prpf31 can interact with and sequester wild-type Prpf31 or other spliceosomal components

  • Example finding: In mouse models, mutant A216P Prpf31 protein attracted wild-type Prpf31 to cytoplasmic aggregates, depleting functional Prpf31 from the nucleus—a classic dominant-negative mechanism

• Splicing activity assays:

  • Approach: Measure splicing efficiency of reporter constructs in the presence of wild-type or mutant Prpf31

  • Results: AD5-expressing retinas showed disrupted mRNA splicing of key photoreceptor genes (rho, gnat1, guk1, rcv1, calb2) with significantly stabilized introns, indicating active interference with splicing—consistent with dominant-negative effects

• Compensation response analysis:

  • Technique: Measure endogenous prpf31 expression after introduction of mutations

  • Observation: AD5 mutant mRNA injection resulted in increased endogenous prpf31 expression, suggesting a compensatory response to functional deficiency, whereas SP117 caused downregulation of endogenous prpf31

This multi-faceted approach has revealed that distinct PRPF31 mutations operate through different pathomechanisms, with important implications for therapeutic strategies—haploinsufficiency (like SP117) might be addressed through gene supplementation alone, while dominant-negative mutations (like AD5) may require strategies to neutralize the toxic mutant protein.

What are the best methods for producing and purifying recombinant Danio rerio Prpf31 for biochemical and structural studies?

For researchers requiring high-quality recombinant Danio rerio Prpf31 for biochemical and structural studies, several expression systems and purification strategies can be employed:

• Expression Systems Comparison:

  Expression System	Advantages	Considerations	Recommended for
  E. coli	High yield, cost-effective, rapid	May lack post-translational modifications; potential folding issues	Domain studies, antibody production, protein-RNA interaction studies
  Yeast (S. cerevisiae/P. pastoris)	Eukaryotic folding machinery, moderate yield	More complex than bacterial systems; longer production time	Full-length functional studies requiring proper folding
  Baculovirus/insect cells	Excellent for complex eukaryotic proteins; proper folding	Higher cost; specialized expertise required	Structural studies; protein-protein interaction analyses
  Mammalian cells	Most native-like post-translational modifications	Highest cost; lowest yield	Studies requiring mammalian-specific PTMs or maximum functionality

• Optimization strategies for Prpf31 expression:

  • Codon optimization: Adapt codon usage for the expression system

  • Fusion tags: N-terminal His6, GST, or MBP tags can improve solubility

  • Expression temperature: Lower temperatures (16-18°C) often improve proper folding

  • Truncated constructs: Express functional domains separately if full-length protein proves challenging

• Purification workflow:

  • Initial capture: Affinity chromatography (Ni-NTA for His-tagged constructs)

  • Intermediate purification: Ion exchange chromatography (typically anion exchange as Prpf31 has theoretical pI ~5.5)

  • Polishing step: Size exclusion chromatography to ensure homogeneity

  • Tag removal: Site-specific protease cleavage (TEV or PreScission)

  • Quality control: SDS-PAGE, Western blot, and mass spectrometry analysis

• Protein activity validation:

  • RNA binding assays: Electrophoretic mobility shift assays (EMSA) with U4 snRNA

  • Tri-snRNP assembly assays: In vitro reconstitution with other spliceosome components

  • Thermal stability assessment: Differential scanning fluorimetry

  • Structure validation: Circular dichroism spectroscopy for secondary structure analysis

• Considerations for structural studies:

  • Crystallization: Screen for crystallization conditions with and without RNA partners

  • Cryo-EM: Consider Prpf31 in complex with other spliceosomal components

  • NMR: Isotope labeling strategies for specific domain structural analysis

• Special considerations for Danio rerio Prpf31:

  • The full-length protein contains 508 amino acids with functional domains including Nop and Prp31 C-terminal domains

  • Commercial recombinant Danio rerio Prpf31 is available with purity ≥85% as determined by SDS-PAGE

  • For maximum yield, consider expressing the 29 kDa Nop domain separately for RNA-binding studies

Successful expression and purification of Danio rerio Prpf31 enables diverse applications including structural studies, functional assays, protein-RNA interaction studies, and the generation of research tools such as antibodies for investigating splicing mechanisms in zebrafish models.

How does the mechanism of retinal degeneration in zebrafish Prpf31 models compare to mammalian models?

The mechanisms of retinal degeneration in zebrafish Prpf31 models compared to mammalian models reveal important similarities and differences that inform our understanding of PRPF31-associated retinal diseases:

• Phenotypic manifestation differences:

  Feature	Zebrafish Prpf31 Models	Mouse Prpf31 Models	Human PRPF31 Disease
  Onset of degeneration	Rapid in knockout/mutant models	Delayed or absent in heterozygous knockouts	Typically second to third decade of life
  Primary affected cells	Primarily photoreceptors	RPE pathology precedes photoreceptor degeneration in some models	Predominantly rod photoreceptors initially
  Phenotype in heterozygotes	Clear retinal defects in morphants and transgenic models	Variable; some models show no obvious retinal phenotype until aged	Incomplete penetrance with variable expressivity

• Molecular mechanism commonalities:

  • Both zebrafish and mammalian models demonstrate the critical role of PRPF31 in pre-mRNA splicing

  • Aberrant splicing of retina-specific transcripts is observed across species

  • The ubiquitous expression but retina-specific pathology pattern is consistent

• Haploinsufficiency versus dominant-negative mechanisms:

  • Zebrafish models clearly demonstrate both mechanisms depending on the mutation:

    • SP117 operates through haploinsufficiency

    • AD5 displays dominant-negative characteristics

  • Mouse models have shown mixed results:

    • Initial heterozygous knockout mice showed no retinal phenotype, questioning the haploinsufficiency model

    • The A216P knock-in mouse model showed evidence of dominant-negative effects with mutant Prpf31 attracting wild-type protein to cytoplasmic aggregates

• Developmental versus degenerative processes:

  • Zebrafish models often show developmental defects in addition to degenerative phenotypes

  • Mammalian models typically exhibit more age-related progressive degeneration

  • This difference may reflect the rapid development and shorter lifespan of zebrafish

• Response to therapeutic interventions:

  • Gene augmentation studies in both zebrafish and mice have shown promising results:

    • AAV-mediated PRPF31 gene augmentation prevented retinal degeneration in CRISPR-generated mouse models

    • Similar approaches in zebrafish rescued structural and functional defects

• Species-specific insights:

  • Zebrafish provide advantages for high-throughput screening and early development studies

  • Mouse models better recapitulate the chronic progressive nature of human disease

  • The complementary use of both models provides a more complete understanding of disease mechanisms

• Involvement of non-retinal tissues:

  • Recent zebrafish studies have uncovered unexpected roles for Prpf31 in hematopoietic stem and progenitor cell (HSPC) expansion

  • Mouse models with complete Prpf31 knockout show embryonic lethality similar to zebrafish

  • These findings highlight the essential nature of Prpf31 beyond the retina, though clinical manifestations in humans remain predominantly ocular

These comparative insights suggest that while basic pathomechanisms are conserved across species, the manifestation and progression of retinal degeneration may vary due to species-specific factors in retinal biology, development, and aging processes.

What are the latest advances in gene therapy approaches for PRPF31-associated retinal diseases based on zebrafish and mouse models?

Recent advances in gene therapy approaches for PRPF31-associated retinal diseases represent a promising frontier in translational research, with significant developments emerging from zebrafish and mouse models:

• Proof-of-concept for AAV-mediated gene augmentation:

  • A groundbreaking 2022 study demonstrated the first in vivo proof-of-concept for AAV-mediated gene therapy to treat PRPF31-retinitis pigmentosa

  • Using a CRISPR/Cas9 knockout mouse model with rapid retinal degeneration, researchers showed that AAV-mediated human PRPF31 gene augmentation:

    • Prevented the formation of retinal pigmentation

    • Preserved normal retinal structure including photoreceptors and RPE

    • Maintained retinal function

    • Downregulated cell death and gliosis markers

• Vector optimization strategies:

  • Promoter selection: The ubiquitous CAG promoter has been successfully used in mouse models

  • Vector capacity considerations: PRPF31 cDNA (~1.6 kb) fits well within AAV packaging limits

  • Serotype selection: AAV8 and AAV9 have shown effective retinal transduction in preclinical models

• Timing of intervention insights:

  • Rescue studies in rapidly degenerating zebrafish models highlighted the importance of early intervention

  • Encouraging results from mouse models where gene augmentation prevented degeneration in a disease model with more rapid progression than human patients suggests potential efficacy even in established disease

• Disease mechanism-specific approaches:

  • For haploinsufficiency mutations (like SP117), simple gene supplementation appears sufficient

  • For dominant-negative mutations (like AD5), approaches may require:

    • Higher doses of wild-type PRPF31 to overcome toxic effects of mutant protein

    • Potential combination with strategies to neutralize the mutant protein

• Delivery route optimization:

  • Subretinal injections provide targeted delivery to photoreceptors and RPE

  • Intravitreal approaches may offer broader retinal coverage with less invasive procedures

  • Suprachoroidal delivery is being explored as an alternative route

• Cross-species validation:

  • Successful demonstrations in both zebrafish and mouse models strengthen the translational potential

  • Recent work has extended AAV-CRISPR/Cas9-PRPF31 knockout constructs to human and non-human primate retinal explants, laying a foundation for primate models

• Therapeutic window considerations:

  • Zebrafish studies with the AD5 mutation revealed a critical period before photoreceptor degeneration becomes irreversible

  • Mouse studies suggest gene augmentation can be effective even in models with more severe degeneration than typically seen in patients

• Challenges and future directions:

  • Addressing incomplete penetrance and variable expressivity characteristic of PRPF31-RP

  • Development of inducible or regulated expression systems to optimize therapeutic levels

  • Investigation of combination approaches targeting both RNA splicing rescue and retinal neuroprotection

These advances represent significant progress toward clinical translation, with animal models providing critical insights into optimal therapeutic strategies for PRPF31-associated retinal diseases. The successful demonstration of gene augmentation efficacy in rapidly degenerating models suggests particular promise for treating the typically slower progression seen in human patients.

How can researchers accurately assess the functional impact of novel PRPF31 variants identified in patients?

Accurately assessing the functional impact of novel PRPF31 variants requires a comprehensive multi-level approach combining in silico prediction, in vitro biochemical characterization, and in vivo modeling:

• In silico prediction and structural analysis:

  • Domain impact assessment: Determine if the variant affects functional domains (Nop domain, Prp31 C-terminal domain)

  • Conservation analysis: Evaluate evolutionary conservation across species

  • Protein structure modeling: Predict effects on protein folding and stability

  • Splicing prediction algorithms: Assess potential impact on PRPF31 pre-mRNA splicing

• In vitro functional characterization:

  • Protein stability assessment:

    • Express wild-type and variant proteins in cell culture

    • Monitor protein levels over time with cycloheximide chase assays

    • Compare degradation rates as seen with SP117 (rapidly degraded) vs. AD5 (moderately stable)

  • Subcellular localization analysis:

    • Use fluorescently tagged constructs to track localization

    • Compare with known patterns (SP117: cytoplasmic mislocalization; AD5: initial nuclear localization followed by cytoplasmic accumulation)

  • Protein-protein interaction studies:

    • Assess interaction with key spliceosomal components (PRPF6, U4 snRNA)

    • Determine if variant affects tri-snRNP assembly

    • Test whether variant protein interacts with wild-type PRPF31 (potential dominant-negative mechanism)

  • Splicing efficiency assays:

    • Use minigene constructs to measure splicing activity

    • Assess impact on splicing of retina-specific transcripts

    • Examine intron retention patterns in known target genes

• Zebrafish in vivo modeling:

  • mRNA injection approach:

    • Synthesize mRNA encoding the variant

    • Inject into 1-cell stage zebrafish embryos

    • Assess embryonic development, survival rates, and phenotypes

    • Compare to effects of known pathogenic variants (SP117, AD5)

  • CRISPR/Cas9 knock-in strategy:

    • Generate precise mutations mimicking patient variants

    • Assess retinal structure and function

    • Evaluate photoreceptor morphology and survival

  • Rescue experiments:

    • Test whether variant can rescue prpf31 morphant phenotypes

    • Co-inject with MO to test for dominant-negative effects

    • Compare with known haploinsufficient and dominant-negative mutations

• Mammalian cell culture models:

  • Patient-derived iPSCs differentiated to retinal cells/organoids:

    • Generate photoreceptors carrying the variant

    • Assess cell morphology, survival, and function

    • Examine transcriptome and splicing patterns

  • CRISPR-edited cell lines:

    • Introduce variants in RPE or photoreceptor cell lines

    • Analyze splicing efficiency and cell health

• Standardized classification system:

  • Develop a comprehensive scoring system that integrates:

    • In silico predictions

    • Protein stability/localization data

    • Splicing activity results

    • Dominant-negative vs. haploinsufficiency indicators

    • In vivo phenotypic severity

This multi-modal approach is exemplified by studies on the SP117 and AD5 variants, where zebrafish modeling revealed distinct cellular mechanisms (haploinsufficiency vs. dominant-negative) despite both causing retinitis pigmentosa in humans . Such thorough characterization is essential for accurate variant classification, genetic counseling, and selection of appropriate therapeutic strategies.

What molecular mechanisms link Prpf31 deficiency to alternative splicing defects in photoreceptors?

The molecular mechanisms linking Prpf31 deficiency to alternative splicing defects in photoreceptors involve multiple interconnected pathways that collectively compromise photoreceptor-specific gene expression:

• Disruption of tri-snRNP assembly:

  • Prpf31 is essential for the interaction between U5 and U4/U6 snRNPs to form tri-snRNPs

  • Deficiency leads to accumulation of U4/U6 di-snRNPs in Cajal bodies while U5 snRNPs remain in nucleoplasmic speckles

  • This disruption impairs spliceosome assembly and activation, particularly affecting genes with complex splicing patterns

• Selective vulnerability of photoreceptor-specific transcripts:

  • Transcriptome analysis of AD5-expressing zebrafish retinas revealed selective disruption of phototransduction-related genes:

    • rhodopsin (rho)

    • G protein transducin alpha subunit (gnat1)

    • guanylate kinase (guk1)

    • recoverin (rcv1)

    • calbindin 2 (calb2)

  • These transcripts exhibited significantly stabilized introns, indicating specific splicing defects

• Intron retention mechanisms:

  • Prpf31 deficiency leads to widespread intron retention, particularly affecting genes with:

    • Weak splice sites

    • Complex splicing patterns

    • High transcriptional activity (characteristic of photoreceptor genes)

  • Retained introns can trigger nuclear detention of transcripts, preventing their translation

• Nuclear accumulation of incompletely spliced transcripts:

  • Light regulation studies suggest that spliceosome activity modulates nuclear detainment of intron-retained transcripts

  • In Prpf31-deficient cells, this process may be dysregulated, leading to nuclear accumulation of essential photoreceptor transcripts

• Splicing stress response activation:

  • Prpf31 mutations can trigger cellular stress responses:

    • Unfolded protein response activation (observed in Prpf31+/A216P mice with upregulated Hsp70)

    • R-loop formation at sites of transcription-splicing coupling

    • Potential activation of RNA surveillance mechanisms

• Alternative splicing pattern shifts:

  • Photoreceptors employ specialized alternative splicing programs

  • Prpf31 deficiency can shift these patterns, affecting:

    • Exon inclusion/skipping ratios

    • Alternative 5' and 3' splice site selection

    • Use of alternative promoters or polyadenylation sites

• dsRNA accumulation and immune activation:

  • Splicing defects can lead to double-stranded RNA (dsRNA) formation

  • dsRNA can trigger innate immune responses that may contribute to photoreceptor stress and death

• Cell-cycle dysregulation in retinal progenitors:

  • Recent studies in zebrafish Prpf31 knockout models revealed effects on mitosis-related gene splicing

  • This suggests that Prpf31 deficiency may also affect retinal development through cell-cycle regulation

• Differential impacts based on mutation type:

  • Haploinsufficiency mutations (like SP117) reduce total functional Prpf31 below a critical threshold

  • Dominant-negative mutations (like AD5) actively interfere with splicing machinery, potentially through:

    • Competition for binding partners

    • Formation of non-functional complexes

    • Sequestration of other splicing factors