CSTF77 Antibody

What is CSTF77 and what cellular functions does it regulate?

CSTF77 (also known as CSTF3, CF-1 77 kDa subunit, or CstF-77) functions as one of the multiple factors required for polyadenylation and 3'-end cleavage of mammalian pre-mRNAs . It plays a crucial role in the cleavage stimulation factor (CstF) complex that recognizes GU-rich downstream sequence elements during 3' end processing. Research has demonstrated that CSTF77 expression levels significantly impact global alternative polyadenylation patterns and are particularly critical for expression of genes involved in cell cycle regulation . The protein participates in an autoregulatory feedback mechanism to control its own expression levels, similar to its Drosophila homolog su(f) . This autoregulation appears to be part of a broader cellular mechanism that coordinates RNA processing during developmental transitions and cellular differentiation.

What are the available CSTF77 antibody types and their validated applications?

Commercially available CSTF77 antibodies include rabbit polyclonal antibodies suitable for various research applications. The Abcam antibody (ab74407) has been validated for immunohistochemistry on paraffin-embedded samples (IHC-P) from both human and mouse tissues . This antibody was generated using a synthetic peptide corresponding to human cleavage stimulation factor subunit 3 within the amino acid region 650 to the C-terminus . Validated applications include:

The antibody has been successfully used in formalin-fixed, paraffin-embedded human prostate carcinoma tissue and mouse teratoma tissue with DAB detection methods .

How can I verify CSTF77 antibody specificity in my experimental system?

Verifying antibody specificity is critical for reliable research outcomes. For CSTF77 antibody validation, consider these methodological approaches:

• Knockdown controls: Perform siRNA-mediated knockdown of CSTF77 and confirm reduced signal by your detection method. Research has demonstrated that CSTF77 knockdown leads to decreased protein levels detectable by immunoblotting approximately 16 hours after siRNA transfection .

• Tissue/cell type verification: Compare CSTF77 expression patterns across tissues with known differential expression. Research shows varying CSTF77 expression levels across different mouse tissues and human cell lines .

• Isoform detection: Verify whether your antibody detects both full-length CSTF77 (CSTF77.L) and shorter isoforms (CSTF77.S). The abundance of different isoforms varies significantly in different cellular contexts and can impact experimental interpretation .

• Western blot analysis: Confirm the detection of a single band at the expected molecular weight (approximately 77 kDa) without non-specific bands.

How does CSTF77 expression level affect alternative polyadenylation patterns genome-wide?

CSTF77 expression levels have a significant impact on alternative polyadenylation (APA) patterns across the genome. Research using 3'READS (3' region extraction and deep sequencing) analysis following CSTF77 knockdown in C2C12 cells has revealed:

• CSTF77 depletion causes significant changes in APA patterns for numerous genes, with distinct sets showing either upregulation of distal or proximal polyadenylation sites .

• The global 3'UTR length correlates with the ratio of short to long CSTF77 isoforms (CSTF77.S/CSTF77.L) across different tissues and cell types, with a correlation coefficient (R²) of 0.61 in human cells/tissues and 0.71 in mouse cells/tissues .

• CSTF77 knockdown affects genes with specific sequence features around their polyadenylation sites. Downregulated isoforms tend to have enrichment of certain pentamer sequences, while upregulated isoforms show different sequence preferences .

• There is significant overlap between genes showing 3'UTR changes after CSTF77 knockdown and those affected during C2C12 cell differentiation, suggesting CSTF77 is a key regulator of APA during developmental transitions .

This research indicates that CSTF77 functions as a critical regulator of polyadenylation site selection, with its expression level serving as a mechanism to coordinate global RNA processing during cellular state changes.

What is the relationship between CSTF77 auto-regulation and cellular differentiation programs?

CSTF77 participates in a sophisticated auto-regulatory feedback loop that appears integrated with broader cellular differentiation programs:

• Auto-regulatory mechanism: CSTF77 protein regulates the usage of its own intronic polyadenylation site (In3 pA). When CSTF77 protein levels are high, usage of this intronic site increases, producing short isoforms (CSTF77.S) that do not encode functional protein, effectively reducing full-length CSTF77 expression .

• Differentiation dynamics: During C2C12 myoblast differentiation, the ratio of CSTF77.S/CSTF77.L gradually increases, corresponding with decreasing CSTF77 protein levels. After 4 days of differentiation, CSTF77 protein decreases by approximately 46% compared to proliferating cells .

• Global impact on gene expression: CSTF77 knockdown significantly affects the expression of 1,776 genes, with cell cycle-related genes being most significantly downregulated . This suggests that modulation of CSTF77 levels helps cells transition from proliferation to differentiation states.

• Coordination with splicing machinery: The regulation of CSTF77's intronic polyadenylation is influenced by components of the splicing machinery, including U1 snRNP, suggesting coordination between splicing and polyadenylation during differentiation .

These findings demonstrate that CSTF77 auto-regulation is not merely a homeostatic mechanism but an integral component of cellular differentiation programs, potentially linking RNA processing regulation to cell fate decisions.

How do CSTF77 interactions with other cleavage and polyadenylation factors influence experimental outcomes?

CSTF77 functions within a complex network of cleavage and polyadenylation factors, and these interactions significantly influence experimental outcomes:

• Interdependence with CstF-64: CstF-64 (another component of the CstF complex) knockdown affects the CSTF77.S/CSTF77.L ratio, indicating coordinated regulation between these factors . CLIP-seq analysis shows differential CstF-64 binding around polyadenylation sites of transcripts upregulated versus downregulated after CSTF77 knockdown .

• Interaction with other C/P factors: Knockdown experiments of other cleavage and polyadenylation factors, including CFI-25, CFI-68, CFI-59, CPSF-160, and CPSF-73, demonstrate varying effects on CSTF77 isoform ratios, revealing complex regulatory relationships .

• Coordination with splicing machinery: Components of the U1 and U2 snRNPs influence CSTF77 intronic polyadenylation. Knockdown of U1-70K, SF3B1, and U2AF65 affects the CSTF77.S/CSTF77.L ratio, suggesting competition between splicing and polyadenylation machineries .

When designing experiments involving CSTF77, researchers should consider these interactions and potentially include measurements of other RNA processing factors to accurately interpret results. For example, experiments manipulating CSTF77 levels should monitor changes in related factors like CstF-64 to account for potential compensatory mechanisms or cooperative effects.

What optimization strategies are recommended for CSTF77 antibody in immunohistochemistry?

For optimal results using CSTF77 antibody in immunohistochemistry on paraffin-embedded samples (IHC-P), consider the following methodological guidelines:

When assessing results, remember that CSTF77 expression varies across tissues and cell types, with differential expression of isoforms potentially impacting staining patterns.

How can I effectively monitor CSTF77 isoform expression in experimental systems?

Monitoring CSTF77 isoform expression requires specific methodological approaches to distinguish between full-length (CSTF77.L) and shorter isoforms (CSTF77.S):

• RT-qPCR strategy: Design primers that specifically differentiate between CSTF77.L and CSTF77.S isoforms. For measuring CSTF77.S, primers spanning the junction between exon 2 and intron 3 (for isoform 3) or between exon 2 and exon 4 (for isoform 2) are effective .

• Semi-quantitative RT-PCR: This approach can help determine the relative abundance of different CSTF77.S isoforms. Research indicates isoform 2 (without intron 2 retention) is approximately 2-3 fold more abundant than isoform 3 in HeLa and C2C12 cells .

• Reporter constructs: Reporter systems like pRinG-77S can be valuable for monitoring intronic polyadenylation in different experimental conditions. These constructs contain relevant segments of the CSTF77 gene linked to fluorescent reporters, allowing visualization of isoform production .

• Microarray or RNA-seq analysis: For global analysis, use microarray probes or RNA-seq to compare read densities for regions specific to different isoforms. This approach has been used to calculate CSTF77.S/CSTF77.L ratios across different tissues .

How should I interpret contradictory CSTF77 antibody results between transcript and protein levels?

Researchers frequently encounter discrepancies between CSTF77 transcript and protein levels, which can be attributed to several biological mechanisms:

• Auto-regulatory feedback: CSTF77 regulates its own expression through alternative polyadenylation, creating a complex relationship between mRNA isoforms and protein levels . High CSTF77 protein levels promote intronic polyadenylation, increasing CSTF77.S transcripts that produce little to no functional protein .

• Post-transcriptional regulation: Research indicates that certain CSTF77 isoforms encode proteins that undergo rapid degradation. For example, isoform 2 contains coding sequences from intron 3 that may cause protein instability .

• Methodological considerations: When detecting CSTF77 protein from intronic polyadenylation isoforms, studies using various antibodies against the N-terminal region failed to detect expected protein products . Similarly, RFP-tagged constructs expressing intronic isoforms did not yield detectable protein by immunoblotting .

To resolve such discrepancies:

• Employ multiple antibodies targeting different epitopes

• Use complementary detection methods (Western blot, IHC, immunofluorescence)

• Include appropriate controls for transcript-specific detection

• Consider pulse-chase experiments to assess protein stability

Understanding the biological basis for these discrepancies is crucial for correct data interpretation, as they reflect the complex regulatory mechanisms governing CSTF77 expression rather than technical artifacts.

What are the critical controls needed when studying CSTF77 function using antibody-based methods?

When investigating CSTF77 function using antibody-based methods, include these critical controls:

• Knockdown validation: siRNA-mediated knockdown of CSTF77 provides essential negative controls. Research shows CSTF77 protein levels begin decreasing approximately 16 hours after siRNA transfection . Both protein and transcript levels should be monitored, as they follow different depletion kinetics.

• Isoform-specific controls: When targeting specific CSTF77 isoforms, verify the specificity of your approach. For example, knockdown of CSTF77.S mRNAs (shorter isoforms) does not affect CSTF77.L (full-length) expression, while knockdown of CSTF77.L affects both protein levels and CSTF77.S expression .

• Overexpression controls: Exogenous CSTF77 expression should increase endogenous CSTF77.S mRNAs while decreasing CSTF77.L mRNAs if the autoregulatory mechanism is functioning properly .

• Tissue/developmental controls: Different tissues and developmental stages show varying CSTF77 isoform ratios. For example, the CSTF77.S/CSTF77.L ratio changes during cell differentiation . Including appropriate tissue controls can provide context for your experimental system.

• Functional readouts: Include measures of CSTF77-dependent processes, such as alternative polyadenylation patterns of known target genes, to confirm functional consequences of CSTF77 manipulation.

These controls help distinguish between direct effects of CSTF77 manipulation and secondary consequences or technical artifacts.

How can CSTF77 antibody research contribute to our understanding of RNA processing disorders?

CSTF77 antibody research provides crucial insights into RNA processing disorders through several mechanisms:

• Global impact on gene expression: CSTF77 knockdown significantly affects expression of 1,776 genes, with cell cycle-related genes being most prominently downregulated . This widespread impact suggests that CSTF77 dysregulation could contribute to disorders involving aberrant cell proliferation or differentiation.

• Developmental regulation: The correlation between CSTF77 isoform ratios and global 3'UTR length changes during development and differentiation suggests that CSTF77 plays a key role in coordinating developmental RNA processing programs . Disruption of these programs could contribute to developmental disorders.

• Interconnected RNA processing networks: CSTF77 function is integrated with both polyadenylation and splicing machinery, as evidenced by interactions with U1 snRNP components . This positions CSTF77 at a critical intersection of RNA processing pathways implicated in numerous neurological and muscular disorders.

• Cell-type specific expression patterns: Different tissues show varying CSTF77 isoform ratios , suggesting tissue-specific requirements for CSTF77 function that could explain tissue-selective manifestations of RNA processing disorders.

By elucidating these mechanisms, CSTF77 antibody research contributes to our understanding of how RNA processing contributes to human disease and potentially identifies new therapeutic targets for disorders involving aberrant RNA processing.

What future research directions should be prioritized in CSTF77 antibody applications?

Several key research directions would significantly advance our understanding of CSTF77 biology:

• Development of isoform-specific antibodies: Current antibodies may not effectively distinguish between different CSTF77 isoforms or detect potential protein products from shorter isoforms . Developing isoform-specific antibodies would enable more precise characterization of CSTF77 expression patterns.

• Single-cell analysis of CSTF77 expression: Investigating CSTF77 isoform expression at the single-cell level could reveal how heterogeneity in CSTF77 regulation contributes to cell fate decisions during development and differentiation.

• Structure-function relationships: Further investigation of how CSTF77 structure relates to its function in the cleavage stimulation factor complex would enhance our understanding of polyadenylation regulation and potentially identify targets for therapeutic intervention.

• Tissue-specific knockout models: Generating tissue-specific CSTF77 knockout or isoform-specific expression models would help elucidate the physiological roles of CSTF77 in different contexts and potentially reveal tissue-specific requirements.

• Integration with other RNA processing pathways: Expanding research on the coordination between CSTF77 and components of other RNA processing pathways, such as the demonstrated interactions with U1 snRNP , would provide a more comprehensive understanding of RNA processing regulation.