CPSF73-II Antibody

What is CPSF73 and why is it significant for RNA processing research?

CPSF73 is a component of the cleavage and polyadenylation specificity factor (CPSF) complex that plays a key role in pre-mRNA 3'-end formation. It functions as an mRNA 3'-end-processing endonuclease, recognizing the AAUAAA signal sequence and interacting with poly(A) polymerase and other factors to facilitate cleavage and poly(A) addition . CPSF73 is also critically involved in histone 3'-end pre-mRNA processing and is required for entering/progressing through S-phase of the cell cycle . Additionally, it participates in the selective processing of microRNAs (miRNAs) during embryonic stem cell differentiation through its interaction with ISY1, highlighting its multifunctional importance in RNA biology .

What types of CPSF73 antibodies are available for research applications?

Several validated CPSF73 antibodies are available for research applications. Commercial options include rabbit polyclonal antibodies such as ab72295 from Abcam, suitable for immunoprecipitation (IP), Western blot (WB), and immunohistochemistry on paraffin-embedded sections (IHC-P) . Another option is the Proteintech antibody 11609-1-AP that targets CPSF3 (alternative name for CPSF73) for WB, IHC, and IP applications . These antibodies have been validated across multiple experimental systems and react with human and mouse samples, providing researchers with reliable tools for CPSF73 detection and characterization .

What is the expected molecular weight and localization pattern for CPSF73?

CPSF73 has a predicted molecular weight of 77 kDa, which aligns with the observed band size in Western blot experiments . When performing immunofluorescence or immunohistochemistry, CPSF73 exhibits predominantly nuclear localization, consistent with its function in nuclear RNA processing . HA-tagged CPSF73 homologs such as RC-74 show exclusive nuclear localization, while some related proteins like RC-68 can be detected in both nuclear and cytoplasmic compartments . This distinct localization pattern serves as an important validation point when evaluating antibody specificity in microscopy-based applications.

How should I optimize Western blot protocols for CPSF73 detection?

For optimal CPSF73 detection by Western blot, follow these methodological guidelines:

• Sample preparation: Use whole cell lysates from appropriate cell lines (HeLa, 293T, NIH3T3 are well-validated)

• Loading amount: Test a gradient of protein amounts (5-50 μg) to determine optimal loading

• Antibody dilution: Start with manufacturer's recommendations (e.g., 1:1000-1:4000 for Proteintech 11609-1-AP)

• Exposure time: Begin with 30-second exposures, as demonstrated in published protocols

The expected result is a clear band at 77 kDa with minimal background, allowing for confident identification of CPSF73 in your experimental samples .

What are the optimal conditions for immunoprecipitation using CPSF73 antibodies?

For successful immunoprecipitation of CPSF73 and associated complexes:

• Buffer selection: The buffer composition significantly impacts which interactions can be detected. For strong interactions (e.g., with CPSF100 and Symplekin), use stringent conditions (50 mM Tris, pH 8.0, 1% NP40, 0.25% deoxycholate, 150 mM NaCl) . For weaker interactions (e.g., with SLBP or Lsm11), use less stringent conditions (50mM Tris, pH 8.0, 0.1% NP-40, 150 mM NaCl) .

• Antibody amount: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate for optimal precipitation efficiency .

• Controls: Include appropriate controls such as IgG from the same species as the primary antibody to identify non-specific binding .

• Sample processing: Be cautious with heat denaturation before IP, as some antibodies (like those against CstF-77 and CPSF-100/73) may not recognize their antigens after boiling, which can affect experimental outcomes .

Research has demonstrated that CPSF73 efficiently co-immunoprecipitates with CPSF100 and Symplekin under stringent conditions, suggesting they form a stable complex in vivo .

How can I validate CPSF73 antibody specificity for my experimental system?

A comprehensive validation approach for CPSF73 antibodies includes:

• Western blot analysis:

  • Confirm single band at expected 77 kDa molecular weight

  • Test in multiple validated cell lines (HeLa, 293T, NIH3T3)

  • Use gradient loading to demonstrate proportional signal intensity

• Knockdown/knockout validation:

  • Compare antibody signal in control vs. CPSF73-depleted samples

  • This approach confirms specificity and provides functional validation

• Cross-reactivity assessment:

  • Test antibody against known close homologs such as CPSF100

  • Some antibodies (e.g., anti-CPSF-100) are known to cross-react with CPSF73 due to sequence similarity

• Immunofluorescence correlation:

  • Compare localization patterns with published data showing predominant nuclear localization

  • Co-stain with other nuclear markers to confirm appropriate subcellular distribution

These methodological steps ensure that experimental observations truly reflect CPSF73 biology rather than non-specific antibody interactions.

How can CPSF73 antibodies be used to investigate protein complexes in RNA processing?

CPSF73 antibodies are powerful tools for dissecting functional protein complexes in RNA processing through several approaches:

• Co-immunoprecipitation (co-IP) analysis: CPSF73 antibodies can efficiently precipitate intact protein complexes, revealing interaction networks. Research has demonstrated that CPSF73, CPSF100, and Symplekin form a stable core complex under stringent co-IP conditions . Additionally, CPSF73 shows interactions with histone mRNA processing factors like SLBP and Lsm11 under less stringent conditions, suggesting mechanistic links between different RNA processing pathways .

• Sequential IP (tandem affinity purification): Using CPSF73 antibodies in combination with antibodies to other processing factors (e.g., CstF components) can identify subcomplexes with distinct functional roles.

• UV cross-linking experiments: CPSF73 antibodies can immunoprecipitate cross-linked protein-RNA complexes, helping identify direct RNA targets of CPSF73-containing complexes . Such experiments have revealed that CPSF73 is part of post-cross-linking 3'-processing complexes with factors like CstF-64 and CstF-77 .

• Differential complex analysis: Comparing CPSF73-associated proteins across different cell types or conditions can reveal context-specific functions of CPSF73-containing complexes.

These approaches collectively provide mechanistic insights into how CPSF73 participates in distinct multiprotein assemblies to regulate RNA processing events.

What methods can identify differential roles of CPSF73 in canonical versus specialized RNA processing?

To distinguish between CPSF73's roles in different RNA processing pathways:

• Pathway-specific knockdown analysis: Depletion of CPSF73 affects both histone and polyadenylated pre-mRNA 3' end formation, whereas depletion of histone-specific factors (SLBP and Lsm11) only affects histone mRNA processing . This differential effect can be measured by:

  • RT-qPCR with primers spanning processing sites

  • 3' RACE to identify processing defects

  • Northern blotting to detect unprocessed precursors

• ChIP analysis: Chromatin immunoprecipitation with CPSF73 antibodies can reveal its recruitment to different gene loci, comparing occupancy at:

  • Polyadenylated gene 3' regions

  • Histone gene 3' ends

  • miRNA processing sites

• Synchronized cell studies: Since histone mRNA processing is cell cycle-regulated, analyzing CPSF73 complexes in synchronized cells can reveal S-phase-specific interactions relevant to histone processing versus constitutive interactions for canonical polyadenylation.

• Immunofluorescence microscopy: Co-staining for CPSF73 and markers of different nuclear bodies (histone locus bodies vs. general RNA processing bodies) can reveal spatial segregation of different CPSF73 functions.

These methodological approaches have demonstrated that CPSF73, CPSF100, and Symplekin are required for both canonical mRNA polyadenylation and specialized histone mRNA 3' end processing .

How can ChIP experiments with CPSF73 antibodies inform our understanding of co-transcriptional RNA processing?

ChIP experiments using CPSF73 antibodies provide valuable insights into co-transcriptional RNA processing through:

• Genome-wide occupancy patterns: ChIP-seq with CPSF73 antibodies reveals the distribution of CPSF73 recruitment across different gene classes. This approach has been successfully implemented alongside antibodies to other factors like CstF50 and RNA Pol II (Rpb3) .

• Temporal recruitment dynamics: ChIP analysis at different time points or in synchronized cells can reveal how CPSF73 recruitment correlates with transcriptional elongation and termination.

• Integration with RNA expression data: Correlating CPSF73 ChIP signals with RNA-seq data can identify relationships between CPSF73 occupancy and processing efficiency.

• Mechanistic insights from co-occupancy: Comparing CPSF73 ChIP with ChIP for other processing factors or RNA Pol II can reveal mechanistic details about assembly of processing complexes.

These approaches collectively build a comprehensive picture of how CPSF73 participates in co-transcriptional processing events across different gene classes and conditions.

What are common technical challenges with CPSF73 antibodies and how can they be addressed?

When working with CPSF73 antibodies, researchers may encounter several technical challenges:

• Epitope accessibility issues:

  • Problem: Some antibodies may fail to recognize CPSF73 after heat denaturation

  • Solution: Use native IP conditions when possible, or validate antibody performance after denaturation steps

• Cross-reactivity with CPSF100:

  • Problem: Due to sequence similarity, antibodies may cross-react between CPSF73 and CPSF100

  • Solution: Verify specificity using samples with known CPSF73/CPSF100 expression levels; note that some antibodies (e.g., anti-CPSF-100) are known to cross-react with CPSF73

• Complex stability during IP:

  • Problem: CPSF73 interactions vary in strength; some complexes may dissociate during stringent washes

  • Solution: Optimize buffer conditions based on target interactions; use stringent conditions (50 mM Tris, pH 8.0, 1% NP40, 0.25% deoxycholate, 150 mM NaCl) for strong interactions and less stringent conditions (50mM Tris, pH 8.0, 0.1% NP-40, 150 mM NaCl) for weaker interactions

• Variable nuclear extraction efficiency:

  • Problem: CPSF73 is predominantly nuclear, requiring efficient nuclear extraction

  • Solution: Optimize nuclear extraction protocols to ensure complete recovery of nuclear CPSF73

Understanding these technical considerations enables more reliable and reproducible results when using CPSF73 antibodies across different experimental applications.

How do I optimize immunohistochemistry protocols for CPSF73 detection in tissue samples?

For successful CPSF73 detection in tissue sections:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative option: Citrate buffer pH 6.0

  • Test both methods to determine optimal retrieval for your specific tissue

• Antibody dilution and incubation:

  • Start with recommended range (1:500-1:2000)

  • Optimize through titration experiments for each tissue type

  • Consider extended incubation times (overnight at 4°C) for improved sensitivity

• Detection system selection:

  • DAB detection has been successfully used in published protocols

  • For dual labeling with other markers, consider fluorescent detection systems

• Validated positive control tissues:

  • Human tissues: placenta, thyroid cancer

  • Mouse tissues: kidney, cerebellum

These methodological optimizations ensure specific and sensitive detection of CPSF73 in tissue samples while minimizing background and non-specific staining.

How should I interpret knockdown phenotypes when studying CPSF73 function in RNA processing?

When analyzing CPSF73 knockdown experiments:

• Expected molecular phenotypes:

  • Disruption of both canonical polyadenylation and histone mRNA 3' end processing

  • Accumulation of unprocessed pre-mRNAs

  • Read-through transcription beyond normal termination sites

• Distinguishing direct versus indirect effects:

  • Compare with knockdowns of other processing factors

  • CPSF73, CPSF100, and Symplekin knockdowns affect both histone and polyadenylated pre-mRNA processing

  • SLBP and Lsm11 knockdowns specifically affect histone mRNA processing

• Cell cycle considerations:

  • CPSF73 is required for entering/progressing through S-phase

  • Analyze cell cycle profiles in CPSF73-depleted cells

  • Distinguish RNA processing defects from secondary effects due to cell cycle arrest

• Rescue experiments:

  • Complementation with RNAi-resistant CPSF73 should restore normal processing

  • Mutant complementation (e.g., endonuclease-deficient mutants) can identify essential functional domains

These analytical approaches help distinguish direct CPSF73 functions from secondary effects and provide mechanistic insights into its role in RNA processing pathways.

What insights can CPSF73 antibodies provide about the integration of different RNA processing pathways?

CPSF73 antibodies have revealed important insights about the interconnection between RNA processing pathways:

• Shared processing machinery: Immunoprecipitation studies with CPSF73 antibodies have demonstrated that a core complex of CPSF73, CPSF100, and Symplekin participates in both canonical polyadenylation and specialized histone mRNA processing . This finding challenged earlier models that proposed entirely separate machineries for these processes.

• Differential complex composition: While the core CPSF73-CPSF100-Symplekin complex participates in both pathways, CPSF73 antibodies have helped identify pathway-specific interactors. For histone mRNA processing, CPSF73 interacts with SLBP and Lsm11 under less stringent IP conditions .

• Functional hierarchy: Knockdown studies monitored with CPSF73 antibodies have revealed that depletion of core components (CPSF73, CPSF100, Symplekin) affects both processing pathways, while depletion of pathway-specific factors (SLBP, Lsm11) only affects their dedicated pathway .

• Regulatory connections: CPSF73 antibodies have helped establish connections between 3' end processing and:

  • Cell cycle regulation (CPSF73 is required for S-phase progression)

  • MicroRNA biogenesis (CPSF73 is required for selective miRNA processing during stem cell differentiation)

These findings highlight CPSF73's central role in coordinating multiple RNA processing pathways and connecting them to broader cellular functions.

How can CPSF73 antibodies contribute to understanding non-canonical functions of CPSF73?

CPSF73 antibodies are valuable tools for investigating emerging non-canonical functions of CPSF73:

• MicroRNA processing: CPSF73 is required for the selective processing of specific microRNAs during embryonic stem cell differentiation . CPSF73 antibodies can help:

  • Identify CPSF73 association with specific pri-miRNA transcripts

  • Map CPSF73 recruitment to miRNA gene loci through ChIP

  • Characterize CPSF73-containing complexes specific to miRNA processing

• Cell cycle regulation: CPSF73 is required for entering/progressing through S-phase . Antibodies can reveal:

  • Cell cycle-dependent changes in CPSF73 localization

  • Post-translational modifications that might regulate CPSF73 activity

  • Cell cycle-specific protein interactions

• Connections to disease processes: CPSF73 antibodies can help investigate potential roles in:

  • Cancer progression (through altered 3' end processing)

  • Neurological disorders (through impacts on specialized nervous system transcripts)

  • Developmental disorders (through disrupted miRNA processing)

These emerging research directions expand our understanding of CPSF73 beyond its canonical role in mRNA 3' end processing, highlighting its integration into broader cellular regulatory networks.

What methodological approaches can best characterize CPSF73's endonuclease activity?

To study CPSF73's endonuclease activity in RNA processing:

• In vitro cleavage assays:

  • Immunopurify CPSF73-containing complexes using validated antibodies

  • Test cleavage activity on synthetic RNA substrates

  • Compare activity on different substrate types (polyadenylation signals vs. histone 3' end signals)

• Structure-function analysis:

  • Generate endonuclease-dead CPSF73 mutants

  • Use CPSF73 antibodies to confirm proper complex formation of mutant proteins

  • Assess functional consequences through complementation of CPSF73 knockdown

• CRISPR-based approaches:

  • Create endogenous CPSF73 mutations in the endonuclease domain

  • Use CPSF73 antibodies to monitor expression levels and localization of mutant proteins

  • Analyze resulting RNA processing defects

• Direct RNA cleavage site mapping:

  • Combine CPSF73 immunoprecipitation with techniques like CLIP-seq

  • Identify precise cleavage sites genome-wide

  • Compare experimental findings with the known consensus sequence (cleavage after the 5'-ACCCA-3' sequence in histone pre-mRNA)

These methodological approaches provide complementary insights into the biochemical mechanisms of CPSF73-mediated RNA cleavage and its regulation in different processing contexts.