CPSF30 Antibody

What is the primary role of CPSF30 in mRNA processing?

CPSF30 is a key subunit of the CPSF complex that plays a crucial role in mRNA 3' processing. Contrary to earlier assumptions that CPSF160 was the primary recognition component, research has demonstrated that CPSF30, along with Wdr33, directly contacts the highly conserved AAUAAA polyadenylation signal in mammalian mRNA . CPSF30-RNA interaction is essential for mRNA 3' processing, particularly through its zinc finger domains 2 and 3 (ZF2 and ZF3) . This interaction facilitates proper pre-mRNA cleavage and polyadenylation, making CPSF30 a critical factor in determining mature mRNA structure and stability.

How should researchers validate the specificity of CPSF30 antibodies?

To validate CPSF30 antibody specificity, researchers should implement a multi-step approach:

• Western blot validation: Confirm detection of the expected molecular weight bands (approximately 30 kDa), noting that CPSF30 often presents as a doublet due to its two isoforms .

• Immunoprecipitation controls: Include both positive controls (lysates from cells known to express CPSF30) and negative controls (cells with CPSF30 knockdown or knockout).

• Cross-reactivity assessment: Test antibody against recombinant CPSF30 and other CPSF complex components to ensure specific recognition.

• UV cross-linking validation: If using the antibody for RNA-binding studies, validate with UV cross-linking experiments comparing wild-type and mutant RNA sequences (AAUAAA vs. AAGAAA) .

• Peptide competition assay: Pre-incubate the antibody with specific CPSF30 peptides to confirm signal elimination in subsequent detection assays.

What sample preparation methods are optimal for CPSF30 detection in different experimental contexts?

Sample preparation strategies should be tailored to the specific experimental context:

For Western blotting:

• Extract proteins using RIPA buffer supplemented with zinc and protease inhibitors to preserve zinc-finger domain integrity

• Include DTT or β-mercaptoethanol in sample buffers to maintain reducing conditions

• Avoid extensive freeze-thaw cycles that may affect epitope recognition

For immunoprecipitation and RNA-binding studies:

• Use stringent immunoprecipitation conditions with multiple washing steps to reduce background

• For nuclear extracts, implement the method described by Chan et al., which yielded consistent CPSF complex purification with all subunits present at comparable levels

• When analyzing CPSF30-RNA interactions, consider RNase I treatment followed by denaturation in 0.5% SDS prior to immunoprecipitation

For immunofluorescence:

• Use paraformaldehyde fixation (4%) for 15 minutes at room temperature

• Consider permeabilization with 0.1% Triton X-100 to access nuclear proteins

How can researchers use CPSF30 antibodies to investigate metal dependency in RNA binding?

CPSF30 exhibits metal-dependent RNA binding properties that can be investigated using antibodies through the following methodological approach:

• Metal chelation experiments: Treat purified CPSF30 with chelating agents (EDTA for zinc, specific iron chelators for Fe-S clusters) before performing RNA binding assays to assess the impact of metal removal.

• Sequential immunoprecipitation: Implement a two-step IP protocol where CPSF30 is first immunoprecipitated under native conditions, then subjected to metal chelation before a second analysis of RNA binding capacity.

• Metal-reconstitution assays: After metal depletion, selectively reconstitute with zinc or iron compounds to determine which metal restores RNA binding activity, as measured by UV cross-linking experiments .

• Spectroscopic analysis with immunopurified protein: Use CPSF30 antibodies to purify the protein for subsequent analysis by X-ray absorption spectroscopy and UV-visible spectroscopy to characterize the 2Fe-2S cluster and zinc content .

Recent findings demonstrate that CPSF30 binds the AAUAAA hexamer through a cooperative, metal-dependent mechanism. Both zinc and the 2Fe-2S cluster contribute to RNA binding, with removal of zinc or both metals completely abolishing binding, while removal of just iron significantly reduces but does not eliminate binding activity .

What are the methodological considerations when using CPSF30 antibodies to study viral infection mechanisms?

When investigating viral infection mechanisms using CPSF30 antibodies, researchers should consider:

• Viral protein co-immunoprecipitation: CPSF30 antibodies can be used to study interactions with viral proteins such as influenza NS1A, which specifically targets CPSF30's ZF2 and ZF3 domains to suppress host mRNA processing .

• Experimental design for NS1-CPSF30 interaction studies:

  • Use agarose beads conjugated with anti-FLAG antibodies for CPSF30 immunoprecipitation

  • Include appropriate controls (PR8 NS1 as negative, TX NS1 as positive control)

  • Verify NS1 and CPSF30 expression levels by Western blot before co-immunoprecipitation

  • Detect interactions using antibodies against epitope tags (FLAG for CPSF30, HA for NS1)

• Mutation analysis: When studying viral protein interactions with CPSF30, consider specific amino acid residues that affect binding. For example, mutations L103F/I106M/P114S/G125D/N139D in H9N2 NS1 restore its ability to interact with CPSF30 .

• Plant viral systems: In plant models like Arabidopsis thaliana, CPSF30 antibodies can help investigate how CPSF30 facilitates turnip mosaic virus (TuMV) infection, with special attention to the different isoforms (CPSF30-L and CPSF30-S) that exhibit distinct subcellular localization and functions .

How should researchers design experiments to differentiate between CPSF30 isoforms using antibodies?

Researchers should implement the following strategies to differentiate between CPSF30 isoforms:

• Antibody selection or generation:

  • Choose antibodies raised against epitopes that are present in all isoforms for total CPSF30 detection

  • Generate isoform-specific antibodies targeting unique regions (e.g., the YTH domain present only in the CPSF30-L isoform in plants)

• Experimental protocol optimization:

  • Use higher resolution SDS-PAGE (12-15%) to effectively separate the closely migrating isoforms

  • Optimize Western blot transfer conditions for small proteins (25-30 kDa)

• Subcellular fractionation combined with immunodetection:

  • In plant systems, CPSF30-S exhibits distinct localization in cytoplasmic granules with P-body markers (AtDCP1 and AtDCP2)

  • CPSF30-L is more nuclear-localized with m6A binding activity

  • Use subcellular fractionation followed by Western blotting with CPSF30 antibodies to distinguish isoform distribution

• Functional validation:

  • Supplement antibody-based detection with recombinant expression of tagged isoforms

  • Analyze differential complex formation by comparing immunoprecipitation results between isoforms

What are common pitfalls when using CPSF30 antibodies in RNA-protein cross-linking experiments?

Researchers frequently encounter these challenges when utilizing CPSF30 antibodies in RNA-protein cross-linking studies:

• Insufficient cross-linking efficiency:

  • Optimize UV exposure time (recommended: 254 nm UV light for 5 minutes at 4°C)

  • Ensure proper RNA-protein ratio in cross-linking reactions

  • Consider using photoactivatable ribonucleoside-enhanced cross-linking (PAR-CLIP) for increased efficiency

• Non-specific RNA binding detection:

  • Include mutant RNA controls (e.g., AAGAAA instead of AAUAAA)

  • Perform stringent RNase I treatment to remove unprotected RNA segments

  • Denature complexes in 0.5% SDS before immunoprecipitation to disrupt non-specific interactions

• Antibody interference with RNA binding sites:

  • Select antibodies targeting epitopes away from the ZF2 and ZF3 domains that directly contact RNA

  • Consider using tagged CPSF30 constructs when direct antibodies interfere with binding

• Metal-dependent artifacts:

  • Preserve both zinc and iron during sample preparation to maintain native RNA binding capacity

  • Include appropriate metal ions in buffers throughout the experimental procedure

• Signal interpretation challenges:

  • Be aware that CPSF30 often presents as a doublet (~30 kDa) in cross-linking experiments, representing two isoforms

  • Use appropriate size markers and controls to distinguish CPSF30 from other cross-linked proteins

How can researchers optimize immunoprecipitation protocols for studying CPSF30 interactions with other CPSF complex components?

To optimize immunoprecipitation protocols for CPSF30 interactions with other CPSF components:

• Cell line selection and expression system:

  • Establish stable HEK293 cell lines expressing Flag-tagged CPSF73 and HA-tagged CPSF30 (full-length or truncated) for efficient complex purification

  • Account for autoregulatory mechanisms that decrease endogenous CPSF30 when exogenous CPSF30 is expressed

• Buffer optimization:

  • Use nuclear extraction buffers containing 20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT

  • Include protease inhibitors and phosphatase inhibitors to preserve protein integrity

  • Consider adding RNase inhibitors if RNA-mediated interactions are relevant

• IP procedure refinement:

  • Implement stringent washing conditions to reduce background while preserving specific interactions

  • Perform sequential IPs using antibodies against different CPSF components to verify complex integrity

  • Elute under native conditions using specific peptides (e.g., Flag peptides)

• Complex validation methods:

  • Confirm complex composition by mass spectrometry and Western blotting

  • Verify that all known CPSF subunits (CPSF160, CPSF100, CPSF73, CPSF30, hFip1, and Wdr33) plus symplekin are present at comparable levels

  • Assess functional integrity through in vitro cleavage and polyadenylation assays

What strategies can resolve inconsistent results when using CPSF30 antibodies across different experimental systems?

When facing inconsistent results with CPSF30 antibodies across experimental systems, implement these resolution strategies:

• Antibody validation across systems:

  • Perform side-by-side Western blot comparison in different cell types/species

  • Validate epitope conservation through sequence alignment when working with CPSF30 from different species

  • Consider using multiple antibodies targeting different CPSF30 epitopes

• Expression level normalization:

  • Quantify endogenous CPSF30 levels across experimental systems

  • Adjust antibody concentrations accordingly for different detection methods

  • Consider stable isotope labeling with amino acids in cell culture (SILAC) for quantitative comparisons

• Sample preparation standardization:

  • Establish consistent nuclei isolation procedures across cell types

  • Standardize buffer compositions, particularly metal ion concentrations

  • Implement identical cross-linking parameters when applicable

• Technical replicate analysis:

  • Perform at least three biological replicates of key experiments

  • Analyze intra-sample and inter-sample variability

  • Use statistical methods appropriate for immunodetection data (non-parametric tests may be required)

• Recombinant protein controls:

  • Include purified recombinant CPSF30 as positive control

  • Use CPSF30 knockout/knockdown samples as negative controls

  • Consider complementation experiments in CPSF30-deficient systems

How can researchers use CPSF30 antibodies to investigate the differential roles of zinc and iron in CPSF30 function?

To investigate the differential roles of zinc and iron in CPSF30 function using antibodies:

• Metal-selective depletion experiments:

  • Perform selective chelation of zinc or iron followed by immunoprecipitation to assess complex integrity

  • Use metal-specific chelators (TPEN for zinc, desferrioxamine for iron)

  • Compare RNA binding activity after selective metal depletion using UV cross-linking

• Site-directed mutagenesis combined with immunodetection:

  • Generate CPSF30 mutants with alterations in zinc-coordinating residues (CCCH domains) or the 2Fe-2S cluster ligands

  • Express these mutants in cells and use CPSF30 antibodies to assess:
    a) Protein stability and expression levels
    b) Subcellular localization
    c) Interaction with other CPSF components
    d) RNA binding capacity

• Structural analysis with purified protein:

  • Use CPSF30 antibodies to immunopurify the protein for X-ray absorption spectroscopy

  • Analyze both zinc and iron coordination environments

  • Compare spectral features before and after RNA binding

• Functional reconstitution assays:

  • Deplete both metals from CPSF30

  • Selectively reconstitute with either zinc or iron

  • Assess functional recovery using in vitro cleavage and polyadenylation assays

Recent research has shown that CPSF30 contains zinc-finger domains and an unexpected 2Fe-2S cluster, with both metal types contributing to RNA binding. Removal of zinc or both metals completely abolishes RNA binding, while removal of just iron significantly reduces but does not eliminate binding activity, suggesting differential roles in CPSF30 function .

What methodological approaches can determine if CPSF30 antibodies affect the protein's RNA binding capacity?

To determine if CPSF30 antibodies affect RNA binding capacity, researchers should:

• Pre-binding interference assay:

  • Pre-incubate CPSF30 with antibodies before adding RNA substrate

  • Compare RNA binding efficiency (via UV cross-linking or EMSA) between antibody-bound and unbound CPSF30

  • Test multiple antibodies targeting different epitopes to map interference

• Competitive binding analysis:

  • Set up reactions with constant CPSF30 and RNA concentrations

  • Add increasing amounts of antibody

  • Monitor displacement using fluorescence anisotropy or electrophoretic mobility shift assays (EMSA)

• Epitope mapping relative to functional domains:

  • Use limited proteolysis to generate CPSF30 fragments

  • Determine which fragments retain antibody binding via Western blot

  • Cross-reference with known functional domains (particularly ZF2 and ZF3)

  • Generate a structural map of antibody binding sites relative to RNA contact points

• Functional recovery with recombinant protein:

  • If antibody interference is detected, confirm by adding excess recombinant CPSF30

  • Observe restoration of function as recombinant protein competes with antibody-bound protein

How should researchers interpret conflicting data between CPSF30 antibody studies and genetic knockout models?

When faced with discrepancies between antibody-based studies and genetic models:

• Comprehensive validation:

  • Verify knockout efficiency at DNA, RNA, and protein levels

  • Confirm antibody specificity through multiple methods including Western blot in knockout cells

  • Assess potential cross-reactivity with related proteins

• Compensation mechanism analysis:

  • Investigate upregulation of functionally related proteins in knockout models

  • Use RNA-Seq and proteomics to identify compensatory pathways

  • Compare acute depletion (RNAi, CRISPR) versus chronic knockout phenotypes

• Isoform-specific effects:

  • Determine if knockout affects all isoforms equally

  • Consider that antibodies may detect multiple isoforms differently

  • In plants, differentiate between CPSF30-L and CPSF30-S isoforms, which have distinct functions

• Cell type and developmental considerations:

  • Evaluate if phenotypes are tissue-specific or developmental stage-dependent

  • Consider that antibody studies may be conducted in systems different from knockout models

  • Assess if compensatory mechanisms differ between acute and chronic loss of CPSF30

• Functional redundancy assessment:

  • Investigate if related proteins can substitute for CPSF30 function in knockout models

  • Perform double knockouts to address redundancy

  • Use antibodies to assess upregulation of potential compensatory proteins

How can CPSF30 antibodies be utilized to investigate viral interference with host mRNA processing?

CPSF30 antibodies offer several methodological approaches to study viral interference with host mRNA processing:

• Temporal analysis of CPSF30-viral protein interactions:

  • Track CPSF30-NS1 interaction during different stages of influenza infection using co-immunoprecipitation

  • Monitor changes in CPSF30 localization during infection using immunofluorescence

  • Assess changes in CPSF30 post-translational modifications during viral infection

• Structure-function mapping of viral interference:

  • Use CPSF30 antibodies in combination with mutant viral proteins to map interaction domains

  • Implement the co-immunoprecipitation protocol described by Kochs et al., using FLAG-tagged CPSF30 and HA-tagged NS1 proteins

  • Include appropriate controls (PR8 NS1 as negative, TX NS1 as positive control)

  • Analyze how specific mutations (e.g., L103F/I106M/P114S/G125D/N139D in NS1) affect CPSF30 binding

• Competition assays:

  • Determine if viral proteins compete with RNA for CPSF30 binding

  • Compare UV cross-linking efficiency of CPSF30 to AAUAAA RNA in the presence/absence of viral proteins

  • Quantify changes in host mRNA polyadenylation during infection

• Cross-species comparative analysis:

  • Apply CPSF30 antibodies to investigate CPSF30-viral protein interactions across different host species

  • Compare binding patterns between mammalian and plant CPSF30 with their respective viral antagonists

What experimental design is recommended for investigating CPSF30 domain-specific functions using antibody-based approaches?

To investigate domain-specific functions of CPSF30:

• Domain truncation analysis:

  • Generate cell lines expressing Flag-tagged full-length or domain-truncated CPSF30 constructs

  • Immunoprecipitate these proteins using anti-Flag antibodies

  • Assess RNA binding capability through UV cross-linking experiments

  • Quantify binding efficiency when specific domains (ZF1-5, ZK) are deleted

• Domain-specific antibody generation and application:

  • Develop antibodies against individual domains (ZF1-5, ZK)

  • Use these antibodies to:
    a) Block specific domains and assess functional consequences
    b) Immunoprecipitate domain-specific interaction partners
    c) Track domain exposure under different cellular conditions

• Functional complementation strategy:

  • Establish CPSF30-depleted cell lines

  • Reintroduce domain mutants (particularly ΔZF2 or ΔZF3)

  • Use antibodies to verify expression levels are comparable to wild-type

  • Assess rescue of RNA processing defects

• Comparative analysis of domains across species:

  • Implement cross-species immunoprecipitation with antibodies recognizing conserved epitopes

  • Compare domain functions between mammalian CPSF30 and plant homologs

Based on previous research, CPSF30-RNA interactions are primarily mediated by ZF2 and ZF3 domains, while the ZK domain contributes to but is not essential for RNA binding. Deletion of either ZF2 or ZF3 does not interfere with CPSF30's association with the CPSF complex but abolishes RNA binding capability .

How can researchers apply CPSF30 antibodies to study its role in plant immune responses and viral susceptibility?

For studying CPSF30's role in plant immunity and viral susceptibility:

• Tissue-specific and stimulus-responsive expression analysis:

  • Use immunohistochemistry with CPSF30 antibodies to map expression patterns in different plant tissues

  • Quantify changes in CPSF30 levels and isoform ratios during pathogen infection

  • Compare CPSF30-L and CPSF30-S expression dynamics during viral infection

• Protein-protein interaction network mapping:

  • Immunoprecipitate CPSF30 from infected vs. uninfected plant tissues

  • Identify differential interaction partners by mass spectrometry

  • Validate key interactions with co-immunoprecipitation and bimolecular fluorescence complementation

• m6A-related function investigation:

  • Use CPSF30 antibodies (particularly against CPSF30-L with m6A binding activity) to study:
    a) m6A reader function during viral infection
    b) Co-localization with other m6A machinery components
    c) Changes in m6A patterns during defense responses

• Subcellular dynamics tracking:

  • Monitor CPSF30 localization changes during infection using immunofluorescence

  • Focus on CPSF30-S coalescence with P-body markers (AtDCP1 and AtDCP2) in cytoplasmic granules

  • Assess relationship between CPSF30 localization and viral replication complexes

Research has demonstrated that CPSF30 significantly facilitates turnip mosaic virus (TuMV) infection in Arabidopsis thaliana, with the CPSF30-L isoform (containing m6A binding activity) emerging as the primary isoform responding to TuMV infection. The CPSF30-S isoform exhibits distinct subcellular localization patterns, suggesting divergent regulatory mechanisms between isoforms .