PRPF4 Antibody

What is PRPF4 and what cellular functions does it perform?

PRPF4 (Pre-mRNA Processing Factor 4) is a 58 kDa spliceosomal protein that plays an essential role in pre-mRNA splicing. It functions as a component of the U4/U6-U5 tri-snRNP complex involved in spliceosome assembly and is also a component of the precatalytic spliceosome (spliceosome B complex) . PRPF4's structure includes a C-terminal WD-40 domain (residues 220-522) and an N-terminal region (residues 1-169) that has been characterized as intrinsically disordered . This protein is critical for the proper functioning of spliceosomes across eukaryotes, with particular importance in retinal tissues where mutations in PRPF4 and other splicing factors can lead to retinitis pigmentosa .

What are the key structural domains of PRPF4 that antibodies typically target?

PRPF4 contains several structurally distinct regions that serve as antibody targets:

• N-terminal region (residues 1-169): An intrinsically disordered region that contains binding sites for other splicing factors, particularly PPIH

• Basic region containing nuclear localization signal: Including the highly conserved arginine at position 192

• C-terminal WD-40 domain (residues 220-522): Important for interaction with PRPF3 and implicated in retinitis pigmentosa mutations

Commercial antibodies have been developed targeting various epitopes, including:

• Regions surrounding Ala278 of human PRPF4 protein (Cell Signaling Technology)

• Amino acids 1-260 (Abbexa)

• Various recombinant fragments within the human PRPF4 sequence (Abcam)

The selection of antibodies targeting specific domains should be guided by the research question, as different domains participate in distinct protein-protein interactions.

How should I select the appropriate PRPF4 antibody for my specific research application?

Selection of PRPF4 antibodies should be guided by:

• Target epitope relevance: For studying PRPF4-PRPF3 interactions, select antibodies targeting the C-terminal domain. For PPIH interactions, choose antibodies recognizing the N-terminal region .

• Application compatibility: Verify antibody validation for your specific application (WB, IP, IF, IHC). For example:

  • For western blotting: Many antibodies show a specific band at 58 kDa

  • For immunofluorescence: Select antibodies validated for nuclear speckle staining patterns

  • For immunoprecipitation: Choose antibodies demonstrated to pull down PRPF4 and its interaction partners

• Species cross-reactivity: Determine if your experimental system requires human-specific antibodies or cross-reactive antibodies for model organisms. Some antibodies react with human, mouse, and rat PRPF4 while others are more species-restricted.

• Clonality considerations: Monoclonal antibodies offer higher specificity but may be sensitive to epitope masking, while polyclonal antibodies provide signal amplification but potentially higher background.

Recommended validation approach: Test new antibodies alongside established ones when possible, and include appropriate negative controls (non-transfected cells, isotype controls) .

What are the expected staining patterns for PRPF4 in different experimental systems?

PRPF4 antibodies should produce the following characteristic patterns:

• Immunofluorescence/ICC:

  • Nuclear localization with distinct speckled pattern typical of splicing factors

  • Co-localization with other splicing components (e.g., EFTUD2)

  • Examples show this pattern in multiple cell lines including HeLa, MCF7, and SiHa cells

• Immunohistochemistry:

  • Nuclear staining in tissue samples

  • Particularly strong staining observed in actively transcribing tissues like cervical carcinoma

• Western blotting:

  • Single predominant band at approximately 58 kDa

  • Expression across multiple human cell lines including HeLa, HT-29, SW480, and MCF-7

The wildtype and mutant PRPF4 proteins (such as R192H variant) show identical nuclear localization patterns, as mutations typically affect protein-protein interactions rather than subcellular localization .

What are the optimal protocols for using PRPF4 antibodies in co-immunoprecipitation experiments?

For successful PRPF4 co-immunoprecipitation experiments:

Protocol framework:

• Cell lysis: Use buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, with protease inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C

• Immunoprecipitation: Add PRPF4 antibody (2-5 μg) to 1 mg of pre-cleared lysate and incubate overnight at 4°C

• Bead capture: Add fresh protein A/G beads and incubate for 2-4 hours at 4°C

• Washing: Perform 4-5 washes with reduced detergent buffer

• Elution: Use SDS sample buffer and heat to 95°C for 5 minutes

Key considerations:

• Include appropriate controls, such as IgG isotype control (as demonstrated in published protocols)

• For studying PRPF4-PRPF3 interactions, gentle washing conditions are crucial as this interaction can be sensitive to detergents

• For tri-snRNP complex studies, consider analyzing co-precipitated snRNAs (U4/U6) by Northern blotting

Example application: Published research demonstrated successful co-IP of PRPF4 with PRPF3 using anti-PRPF3 antibodies to pull down [35S]-labeled, HA-tagged PRPF4, showing that the R192H mutation specifically disrupts this interaction while maintaining other protein interactions (e.g., with PPIH) .

How can I design experiments to investigate PRPF4 mutations and their effect on protein interactions?

Based on published research approaches:

• Expression construct preparation:

  • Generate expression constructs containing wildtype and mutant PRPF4 (e.g., p.R192H variant)

  • Include epitope tags (HA or FLAG) for detection and immunoprecipitation

  • Consider both full-length constructs and domain-specific constructs (N-terminal 1-169, 1-98, 106-169)

• Interaction analysis methods:

  • Co-immunoprecipitation: Express tagged constructs in cells (HEK293/HeLa), immunoprecipitate with antibodies against PRPF4 or interaction partners

  • In vitro binding assays: Use [35S]-labeled proteins produced by in vitro translation

  • Biophysical methods: Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) for quantitative binding parameters

• Functional impact assessment:

  • snRNP integration: Analyze co-precipitation of snRNAs (U4/U6) by Northern blotting

  • Cellular localization: Perform immunofluorescence to assess nuclear localization and co-localization with other splicing factors

  • In vivo functional studies: Consider zebrafish or other model systems for assessing functional consequences

Research example: The p.R192H variant of PRPF4 was demonstrated to:

• Maintain normal nuclear localization

• Specifically disrupt binding to PRPF3 while preserving other interactions

• Prevent stable incorporation into snRNPs

• Result in loss of function in zebrafish rescue experiments

This experimental approach separates protein mislocalization effects from specific interaction defects.

How can I use PRPF4 antibodies to investigate the bi-partite binding mechanism between PRPF4 and PPIH?

The bi-partite binding between PRPF4 and PPIH represents a complex interaction that can be investigated using several specialized approaches:

Experimental strategy:

• Domain-specific binding analysis:

  • Express and purify recombinant PRPF4 fragments (PRPF4 1-169, PRPF4 1-98, PRPF4 106-169)

  • Perform binding assays with purified PPIH using:

    • Isothermal Titration Calorimetry (ITC): Determine binding affinities (reported Kd values: 0.5 μM for PRPF4 1-169; 0.08 μM for PRPF4 106-169)

    • Surface Plasmon Resonance (SPR): Measure binding kinetics for each fragment

• Mutational analysis approach:

  • Introduce point mutations in critical residues in both PRPF4 binding sites

  • F122 mutation has been shown to significantly impact binding

  • W133A mutation in PPIH affects interaction with the proline-rich region of PRPF4

  • Test combinations of mutations to assess cooperativity between binding sites

• Structural characterization:

  • Circular Dichroism (CD) analysis to confirm the intrinsically disordered nature of N-terminal PRPF4

  • Note that the N-terminus of PRPF4 does not adopt secondary structure in the presence of PPIH

Data interpretation guidelines:

What approaches can be used to study the role of PRPF4 mutations in retinitis pigmentosa pathogenesis?

Investigating PRPF4 mutations in retinitis pigmentosa requires specialized experimental approaches:

Multi-tier experimental framework:

• Biochemical interaction studies:

  • Express wildtype and mutant PRPF4 (e.g., p.R192H) in mammalian cells

  • Assess incorporation into tri-snRNP complex using co-immunoprecipitation with antibodies against other components (PRPF31, Sm proteins)

  • Analyze co-precipitation of snRNAs (U4/U6) to evaluate functional integration into snRNPs

• Cellular splicing assays:

  • Utilize splicing reporter constructs to measure splicing efficiency

  • Assess global splicing changes using RNA-seq following PRPF4 knockdown/mutation

  • Focus particularly on retina-specific transcripts with complex splicing patterns

• Retinal cell-specific analyses:

  • Develop retinal organoids or use retinal cell lines expressing PRPF4 mutations

  • Evaluate splicing patterns in photoreceptor cells specifically

  • Assess stress responses and cell viability under various conditions

• In vivo model systems:

  • Zebrafish models have demonstrated that the p.R192H mutation represents a functional null allele

  • Introduce equivalent mutations (e.g., via CRISPR) in mouse models

  • Analyze photoreceptor degeneration patterns and progression

Key findings from published research:

• The p.R192H variant disrupts binding to PRPF3, preventing incorporation into the tri-snRNP

• This represents a functional null allele, suggesting haploinsufficiency as the disease mechanism

• All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP, indicating this particle's crucial importance in retinal physiology

What are common problems encountered when using PRPF4 antibodies and how can they be resolved?

Case example: When studying PRPF4-PRPF3 interactions, the R192H mutant shows dramatically reduced co-precipitation. Confirm this is a true biological effect rather than technical issue by:

• Verifying equal expression of wildtype and mutant proteins

• Demonstrating that the mutant maintains other known interactions (e.g., with PPIH)

• Showing consistent results with reverse co-IP approaches

How should researchers interpret PRPF4 antibody data in the context of studying spliceosomal dynamics?

Interpreting PRPF4 antibody data requires consideration of several contextual factors:

Interpretation guidelines:

• Localization patterns:

  • Nuclear speckled pattern is normal for PRPF4 and other splicing factors

  • Changes in speckle size or number may indicate altered splicing dynamics

  • Co-localization with other spliceosomal proteins (EFTUD2, PRPF3) indicates normal integration

• Interaction data interpretation:

  • PRPF4 exhibits bi-partite binding with PPIH through two distinct regions

  • High-affinity binding occurs through residues 106-169

  • The anchoring site is evolutionarily conserved, indicating functional importance

  • PRPF4-PRPF3 interaction is essential for tri-snRNP assembly

• Mutation impact assessment:

  • Mutations may affect specific interactions while preserving others

  • The R192H mutation specifically disrupts PRPF3 binding while maintaining PPIH interaction and normal localization

  • Changes in snRNA co-precipitation indicate functional defects in spliceosome assembly

• Cross-validation approaches:

  • Compare results using different antibodies targeting distinct epitopes

  • Verify findings using complementary techniques (co-IP, IF, functional assays)

  • Consider both wildtype and mutant PRPF4 expression studies alongside knockdown approaches

Research example: In studies of the R192H PRPF4 variant, immunoprecipitation studies showed that while the protein maintained normal nuclear localization and PPIH binding, it specifically lost PRPF3 interaction. This resulted in failure to incorporate into snRNPs as evidenced by reduced co-precipitation of U4/U6 snRNAs, providing a molecular mechanism for the associated retinitis pigmentosa pathology .

How might PRPF4 antibodies be applied to study tissue-specific splicing regulation?

PRPF4 antibodies offer promising approaches for investigating tissue-specific splicing mechanisms:

Methodological approaches:

• Tissue-specific interaction profiling:

  • Perform PRPF4 immunoprecipitation followed by mass spectrometry across different tissues

  • Compare PRPF4-interacting proteins between retinal tissue and other tissues to identify:

    • Retina-specific interaction partners

    • Differential complex formation in vulnerable tissues

  • Use proximity labeling approaches (BioID, APEX) with PRPF4 antibodies to capture transient interactions

• Chromatin immunoprecipitation (ChIP) approaches:

  • Apply ChIP-seq using PRPF4 antibodies to identify sites of co-transcriptional splicing

  • Compare binding patterns across different tissue types

  • Integrate with RNA-seq data to correlate binding with tissue-specific splicing events

• Single-cell analysis techniques:

  • Develop immunofluorescence approaches to analyze PRPF4 distribution in heterogeneous tissues

  • Combine with RNA-FISH to correlate PRPF4 localization with specific transcript processing

  • Apply to retinal organoids to study cell-type specific effects of PRPF4 mutations

Research implications:

• Understanding why universally expressed splicing factors cause tissue-specific disease (retinitis pigmentosa) remains a fundamental question

• PRPF4 antibodies could help identify tissue-specific cofactors or regulatory mechanisms

• Investigation of the relationship between PRPF4 and retina-specific splicing events may reveal therapeutic targets

What are emerging techniques for studying the intrinsically disordered regions of PRPF4 using antibody-based approaches?

The intrinsically disordered N-terminal region of PRPF4 presents unique challenges and opportunities for antibody-based research:

Advanced methodological approaches:

• Conformation-specific antibody development:

  • Generate and characterize antibodies that recognize specific conformational states of the disordered N-terminal region

  • Apply these to detect binding-induced conformational changes

  • Use epitope mapping to identify regions that undergo disorder-to-order transitions

• In-cell structural biology approaches:

  • Combine PRPF4 antibody-based proximity labeling with mass spectrometry

  • Apply FRET-based approaches with conformation-sensitive PRPF4 antibodies

  • Use antibody-based protection assays to map interaction surfaces in living cells

• Dynamic interaction analysis:

  • Develop antibodies targeting different segments of the bi-partite binding interface

  • Use these to probe the kinetics and sequence of interactions between PRPF4 and partners like PPIH

  • Apply super-resolution microscopy with domain-specific antibodies to track conformational changes during spliceosome assembly

Research context:

• CD spectroscopy has confirmed that PRPF4 1-169, PRPF4 106-169, and PRPF4 1-98 regions are predominantly unstructured

• The N-terminal region does not adopt secondary structure even when bound to PPIH

• This intrinsic disorder may be functionally important for spliceosome assembly dynamics

Studying these intrinsically disordered regions may reveal new therapeutic opportunities, as they often represent sites for small molecule intervention that could modulate protein interactions in diseases like retinitis pigmentosa.