C19orf43 Antibody

What is C19orf43 and why is it significant for telomere biology research?

C19orf43, also known as MGC2803, fSAP18, or TRIR (Telomerase RNA component interacting RNase), is a protein coding gene located on Chromosome 19: p12 that encodes a 176-amino acid protein with a molecular weight of approximately 18.4 kDa (observed at ~28 kDa in Western blots) . It functions as an exoribonuclease in the telomerase RNA 3' end processing complex, capable of cleaving all four unpaired RNA nucleotides from both 5' and 3' ends with higher efficiency for purine bases .

C19orf43 has gained significant research attention due to its:

• Role in telomere cohesion resolution through interaction with tankyrase

• Ability to counteract persistent telomere cohesion in aged cells and ALT cells

• Involvement in TERRA (telomeric repeat-containing RNA) regulation

• Participation in RNA-DNA hybrid (R-loop) formation

Research methodology: To study C19orf43's functions in telomere biology, researchers typically employ knockdown/knockout approaches followed by rescue experiments with wild-type or mutant constructs, coupled with techniques like FISH (Fluorescence In Situ Hybridization), co-immunoprecipitation, and RNA dot blot analysis .

What are the optimal applications for different types of C19orf43 antibodies?

C19orf43 antibodies are valuable tools for multiple molecular and cellular applications. Based on published research and commercial data, here's a methodological guide for their application:

Methodological approach: For accurate results, always validate antibody specificity with appropriate positive and negative controls. When possible, use multiple antibodies raised against different epitopes of C19orf43 to confirm findings .

How can researchers validate the specificity of C19orf43 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For C19orf43 antibodies, implement the following comprehensive validation strategy:

• Genetic validation:

  • Compare signal in wild-type cells versus C19orf43 knockout/knockdown cells

  • Published research shows effective validation using C19orf43 shRNA-expressing lentiviruses (#1 and #4)

• Epitope mapping:

  • Confirm recognition of the intended epitope sequence

  • C19orf43 has several characterized immunogen sequences:

    • Full-length protein (1-176 aa encoded by BC000216)

    • C-terminal region: GSTLSFVGKRRGGNKLALKTGIVAKKQKTEDEVLTSKGDAWAKYMAEVKKYKAHQCGDDDKTR

• Orthogonal validation:

  • Correlate antibody results with mRNA expression data

  • Confirm findings using multiple antibodies targeting different epitopes

• Positive controls:

  • Use recombinant C19orf43 protein

  • Use cell lines with confirmed C19orf43 expression (placenta, 293 cells, A431 cells)

• Cross-reactivity testing:

  • When working with mouse or rat models, verify cross-reactivity (mouse shares 98% sequence identity in some epitope regions)

Methodological note: When using commercial antibodies, review their validation data and consider performing additional validation experiments specific to your experimental system .

How should researchers design experiments to investigate C19orf43's role in telomere cohesion using antibody-based techniques?

Investigating C19orf43's role in telomere cohesion requires sophisticated experimental design combining genetic manipulation, antibody-based detection, and functional assays:

Experimental design framework:

• Genetic manipulation setup:

  • Generate stable C19orf43-depleted cell lines using shRNA (#1 and #4) or CRISPR/Cas9 (KO#2 and KO#4)

  • Create rescue constructs:

    • FLAG-C19orf43 wild-type

    • FLAG-C19orf43 R7G (N-terminal TBM mutant)

    • FLAG-C19orf43 R106G (Second TBM mutant)

• Immunoprecipitation protocol:

  • Use anti-TNKS antibody for co-IP of endogenous C19orf43

  • Use anti-FLAG antibody for FLAG-tagged C19orf43 variants

  • Western blot analysis with C19orf43 antibody (1:1000 dilution)

• Telomere cohesion assessment:

  • FISH analysis using telomere-specific probes

  • Quantify telomere signals as singlets or doublets

  • Compare cohesion patterns between:

    • Control cells

    • C19orf43-depleted cells

    • Rescue with WT or mutant C19orf43

• Cell model considerations:

  • HEK293T cells for standard experiments

  • Pre-senescent WI38 cells for aging studies

  • ALT GM847 cells for alternative lengthening of telomeres context

Research findings indicate that C19orf43.WT and R106G (but not R7G) counteract persistent telomere cohesion, demonstrating that C19orf43's N-terminal tankyrase-binding motif is critical for this function .

What methodological approaches are recommended for using C19orf43 antibodies in RNA-protein interaction studies?

Investigating interactions between C19orf43 and RNA, particularly TERRA, requires specialized methodological approaches:

RNA Immunoprecipitation (RIP) methodology:

• Cell preparation:

  • Transfect cells with FLAG-C19orf43 or FLAG-vector control

  • HEK293T cells show good expression for these experiments

• Cross-linking protocol:

  • Use 1% formaldehyde for protein-RNA crosslinking

  • Quench with glycine (125 mM final concentration)

  • Wash cells with ice-cold PBS

• Immunoprecipitation:

  • Lyse cells in RIP buffer (containing RNase inhibitors)

  • Pre-clear lysate with protein A/G beads

  • Incubate with anti-FLAG antibody (1:100 dilution)

  • Capture complexes with protein A/G beads

  • Perform stringent washes to remove non-specific interactions

• RNA analysis:

  • Reverse crosslinks (65°C incubation)

  • Extract RNA using TRIzol or equivalent

  • Perform qRT-PCR with TERRA-specific primers and controls (GAPDH)

  • Express results as percent input

• R-loop analysis extension:

  • For studying RNA-DNA hybrids, use DNA:RNA immunoprecipitation (DRIP)

  • Utilize S9.6 antibody that detects R-loops

  • Compare results between control and C19orf43-depleted cells

Research finding: C19orf43 associates specifically with TERRA, as demonstrated by enrichment of telomeric sequences in RIP experiments, which is consistent with its role as an exoribonuclease in telomerase RNA processing .

How can researchers optimize C19orf43 antibody usage for detecting low expression levels in telomere-related pathways?

Detecting low-abundance proteins like C19orf43 in telomere-related pathways presents technical challenges that require optimization:

Signal enhancement methodology:

• Sample preparation optimization:

  • Enrich nuclear fractions where C19orf43 is primarily localized

  • Use phosphatase inhibitors to preserve post-translational modifications

  • Consider using detergents optimized for nuclear proteins (e.g., NP-40)

• Western blot sensitivity enhancement:

  • Increase protein loading (50-100 μg total protein)

  • Use PVDF membranes for better protein retention

  • Employ longer primary antibody incubation (overnight at 4°C)

  • Optimal antibody concentrations:

    • 0.04-0.4 μg/mL for standard detection

    • 0.4-1.0 μg/mL for low-abundance detection

• Immunofluorescence signal amplification:

  • Use tyramide signal amplification (TSA)

  • Consider fluorophore-conjugated secondary antibodies with higher quantum yield

  • Recommended dilution range: 0.25-2 μg/mL

  • Use confocal microscopy with appropriate filters

• Co-localization studies:

  • Pair C19orf43 antibody with telomere markers (TRF1, TRF2)

  • Use anti-TNKS antibody to verify interaction at telomeres

  • Employ super-resolution microscopy for detailed co-localization analysis

• Validation in multiple systems:

  • Compare expression across cell lines (placenta, 293 cell, A431 cell lysates show detectable signals)

  • Include age-matched controls when studying senescent cells

  • Consider species-specific optimization (human, mouse, rat reactivity varies)

Research insight: Unlike some exonucleases, C19orf43 does not require divalent cations for its activity, which may influence its detection in different buffer conditions .

What are the technical considerations when using C19orf43 antibodies to study its exoribonuclease activity?

Studying C19orf43's exoribonuclease function requires specific technical approaches to preserve activity and accurately measure enzymatic function:

Exoribonuclease activity assessment protocol:

• Protein purification considerations:

  • Express recombinant C19orf43 with appropriate tag (His or GST)

  • Consider C-terminal tagging to preserve N-terminal tankyrase-binding motifs

  • Purify under native conditions to maintain enzymatic activity

  • Note that the conserved C-terminus is necessary for RNase activity

• Substrate preparation:

  • Use T7-transcribed RNA for initial activity testing

  • Prepare RNA oligonucleotides with different nucleotide compositions

  • Include both 5' and 3' end-labeled substrates to determine directionality

  • Test purine-rich versus pyrimidine-rich substrates (C19orf43 shows higher efficiency for purine bases)

• Activity assay conditions:

  • Test across broad temperature and pH ranges

  • No divalent cations are required for C19orf43 activity

  • Include control RNases with known activity profiles

  • Use RNase inhibitors in negative controls

• Detection methods:

  • Use radiolabeled substrates for highest sensitivity

  • Employ denaturing PAGE for product analysis

  • Consider fluorescent-labeled substrates for real-time monitoring

  • Use mass spectrometry to identify cleavage products

• Inhibition studies:

  • Test small molecule inhibitors

  • Use C19orf43 antibodies to potentially block activity

  • Compare wild-type and mutant C19orf43 (particularly C-terminal mutants)

Research finding: hTRIR (C19orf43) cleaves all four unpaired RNA nucleotides from both 5′ and 3′ ends with higher efficiency for purine bases, consistent with its classification as an exoribonuclease .

How can researchers address data contradictions when using C19orf43 antibodies across different experimental systems?

Addressing contradictory results is crucial for advancing scientific understanding. For C19orf43 research, several methodological approaches can help resolve discrepancies:

Contradiction resolution framework:

• Antibody validation across systems:

  • Test multiple C19orf43 antibodies targeting different epitopes:

    • N-terminal region antibodies

    • C-terminal region antibodies (GSTLSFVGKRRGGNKLALKTGIVAKKQKTEDEVLTSKGDAWAKYMAEVKKYKAHQCGDDDKTR)

    • Full-length protein antibodies (1-176 aa)

  • Verify species cross-reactivity when comparing human, mouse, and rat systems

• Expression level analysis:

  • Quantify C19orf43 expression across different cell types:

    • Different tissues show variable expression levels

    • Apparent contradictions may result from detection threshold differences

  • Use qPCR to correlate protein detection with mRNA levels

• Post-translational modification assessment:

  • Investigate potential phosphorylation or other modifications

  • Use phosphatase treatment prior to Western blot analysis

  • Consider size discrepancy (predicted 18.4 kDa vs. observed ~28 kDa)

• Interaction partner influence:

  • C19orf43 has known interactions with tankyrase and TERRA

  • These interactions may mask antibody epitopes in certain contexts

  • Use detergents or salt concentrations that disrupt protein-protein interactions

• Functional assay standardization:

  • For TERRA level studies, compare RNA dot blot and northern blot results

  • When studying telomere cohesion, standardize FISH analysis parameters

  • For RNase activity, establish consistent substrate and reaction conditions

Research observation: Studies have shown both reduction and potential protection of TERRA by C19orf43, indicating context-dependent functions that require careful experimental design to elucidate.

What methodological approaches should be used to study C19orf43's role in cellular aging using antibody-based techniques?

Cellular aging research requires specialized approaches when studying C19orf43's functions:

Cellular aging research methodology:

• Cell model selection:

  • Pre-senescent WI38 cells provide an established aging model

  • Compare early passage (<30) and late passage (>45) cells

  • Include IMR-90 cells as an alternative fibroblast aging model

  • Consider Werner Syndrome or Hutchinson-Gilford Progeria cells for accelerated aging models

• Senescence marker correlation:

  • Pair C19orf43 antibody detection with:

    • SA-β-galactosidase staining

    • p16^INK4a and p21^CIP1 expression

    • SASP (Senescence-Associated Secretory Phenotype) markers

  • Use immunofluorescence for co-localization studies

• Telomere analysis protocol:

  • Measure TERRA levels using RNA dot blot normalized to 18S rRNA

  • Quantify telomere cohesion using FISH analysis

  • Assess telomere length via qPCR or Southern blot

  • Evaluate telomerase activity using TRAP assay

• Rescue experiment design:

  • Transfect pre-senescent cells with:

    • Vector control

    • C19orf43.WT

    • C19orf43.R7G (non-functional N-terminal TBM mutant)

  • Perform immunoblotting to confirm expression

  • Measure cohesion resolution effectiveness

• Chromosome 19 correlation studies:

  • Investigate co-regulation with other chromosome 19 genes

  • Five tumor suppressor genes on chromosome 19 (SIRT6, BBC3, STK11, CADM4, GLTSCR2) show altered expression in some contexts

  • Use gene expression profiling to identify co-regulated networks

Research finding: C19orf43.WT (but not R7G mutant) counteracts persistent telomere cohesion in aged cells, suggesting its N-terminal tankyrase-binding motif is critical for resolving age-associated telomere cohesion defects .

What is the current understanding of using C19orf43 antibodies in cancer research, and what methodological improvements are needed?

C19orf43's potential role in cancer biology is emerging, with specific methodological considerations for antibody-based studies:

Cancer research methodology framework:

• Cancer cell line profiling:

  • Screen C19orf43 expression across cancer cell panels using validated antibodies

  • Include normal cell counterparts as controls

  • Quantify using Western blot with densitometry analysis

  • Correlate with:

    • Telomerase activity

    • ALT pathway activation

    • TERRA levels

    • Tumor suppressor status

• Patient sample analysis:

  • Optimize immunohistochemistry protocols for FFPE tissues:

    • Recommended dilution: 1:200-1:500

    • Antigen retrieval optimization

    • DAB versus fluorescent detection

  • Compare expression across:

    • Tumor versus adjacent normal tissue

    • Different cancer stages

    • Treatment-resistant versus sensitive tumors

• Functional studies in cancer contexts:

  • Use shRNA or CRISPR/Cas9 to modulate C19orf43 in cancer cells

  • Measure effects on:

    • Proliferation

    • Telomere maintenance

    • Genomic stability

    • DNA damage response

  • Monitor TERRA levels and R-loop formation

• Genomic analysis integration:

  • Correlate with chromosome 19 alterations

  • Note that chromosome 19 deletions affect genes on both p and q arms

  • Five tumor suppressor genes on chromosome 19 show altered expression in cancer contexts

• Methodological improvements needed:

  • Development of phospho-specific antibodies for C19orf43

  • Generation of antibodies against specific functional domains

  • Creation of inducible expression systems for temporal studies

  • Establishment of in vivo models with tissue-specific expression

Research insight: The relationship between C19orf43 and cancer remains an emerging area that requires further investigation, particularly given its role in telomere biology and potential interactions with tumor suppressors on chromosome 19 .

How should researchers design immunoprecipitation experiments to study C19orf43's protein interactions?

Immunoprecipitation is a powerful technique for studying C19orf43's protein interactions, particularly with tankyrase:

Optimized co-immunoprecipitation protocol:

• Cell lysis optimization:

  • Use NP-40 or CHAPS-based lysis buffers (0.5-1%)

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors to preserve phosphorylation states

  • Consider benzonase treatment to remove nucleic acid interference

• Antibody selection strategy:

  • For endogenous interactions:

    • Use anti-TNKS antibody to pull down endogenous C19orf43

    • Detect with C19orf43 antibody in Western blot

  • For tagged protein studies:

    • Use anti-FLAG antibody for FLAG-C19orf43 pulldown

    • Compare WT, R7G, and R106G mutants

• Controls framework:

  • Input sample (5-10% of lysate)

  • IgG control (same species as primary antibody)

  • Blocking peptide competition control

  • Reciprocal IP validation (IP with C19orf43, detect TNKS)

  • C19orf43-depleted cells as negative control

• Washing stringency gradient:

  • Start with standard washing conditions

  • Increase salt concentration (150-500 mM) to test interaction strength

  • Test detergent effects on interaction stability

  • Consider glycerol addition (5-10%) to stabilize complexes

• Detection and quantification:

  • Use appropriate primary antibodies (1:1000-1:5000)

  • Employ HRP-conjugated or fluorescent secondary antibodies

  • Perform densitometry for quantitative analysis

  • Compare interaction efficiency across experimental conditions

Research finding: Endogenous C19orf43 specifically co-immunoprecipitates with endogenous TNKS, and mutation of the N-terminal tankyrase-binding motif (R7G) disrupts this interaction, while the R106G mutation has minimal effect .

What are the best methodological approaches for studying C19orf43's role in TERRA regulation and R-loop formation?

Investigating C19orf43's impact on TERRA levels and R-loop formation requires specialized techniques:

TERRA and R-loop analysis methodology:

• TERRA quantification protocols:

  • RNA dot blot analysis:

    • Probe with 32P-labeled (CCCTAA)4 Telo C probe

    • Normalize to 18S rRNA

    • Compare C19orf43 KO/knockdown versus control cells

  • Northern blot analysis:

    • Use Telo C probe for detection

    • Normalize to 18S rRNA

    • Assess TERRA size distribution changes

  • qRT-PCR approach:

    • Design primers specific to telomeric repeats

    • Include subtelomeric primers for chromosome-specific TERRA

    • Calculate percent input in RIP experiments

• R-loop detection methods:

  • DNA:RNA immunoprecipitation (DRIP):

    • Use S9.6 antibody that detects RNA-DNA hybrids

    • Compare control versus C19orf43-depleted cells

    • Analyze by qPCR with telomere-specific primers

  • Immunofluorescence microscopy:

    • S9.6 antibody staining

    • Co-staining with telomere markers

    • Quantify co-localization events

• Inducible system development:

  • Doxycycline-inducible shRNA for temporal studies:

    • Create stable dox-inducible C19orf43 shRNA cell lines

    • Induce with doxycycline for 48 hours

    • Confirm knockdown by immunoblot

    • Measure TERRA and R-loop changes over time

• Activity modulation experiments:

  • Overexpress wild-type versus mutant C19orf43

  • Compare exoribonuclease-deficient mutants

  • Analyze TERRA stability and half-life

  • Assess R-loop formation dynamics

• TERRA subcellular localization:

  • RNA FISH with telomere probe

  • Co-staining with C19orf43 antibody

  • Z-stack confocal imaging

  • Quantitative analysis of co-localization

Research finding: Surprisingly, C19orf43 depletion resulted in reduced TERRA levels as shown by both RNA dot blot and northern blot analysis, suggesting that rather than degrading TERRA, C19orf43 might play a role in protecting or stabilizing it under certain conditions .

How might antibodies against C19orf43 be utilized in studying its potential roles beyond telomere biology?

While C19orf43's role in telomere biology is established, emerging evidence suggests broader functions:

Expanded research applications:

• RNA metabolism studies:

  • Use C19orf43 antibodies to identify novel RNA substrates:

    • RNA immunoprecipitation followed by sequencing (RIP-seq)

    • CLIP-seq (crosslinking immunoprecipitation with sequencing)

    • Compare substrate profiles across cell types and conditions

  • Investigate processing of non-telomeric RNAs:

    • mRNAs with specific structural features

    • Non-coding RNAs with regulatory functions

    • Viral RNAs as potential substrates

• Stress response investigation:

  • Examine C19orf43 localization changes under stress:

    • Oxidative stress

    • Replication stress

    • Telomere damage

  • Monitor post-translational modifications:

    • Phosphorylation in response to DNA damage

    • Potential ubiquitination or SUMOylation

• Developmental biology applications:

  • Track expression across developmental stages:

    • Embryonic versus adult tissues

    • Stem cell differentiation

    • Tissue regeneration

  • Study knockout phenotypes in model organisms

• Cell cycle regulation exploration:

  • Analyze C19orf43 levels across cell cycle:

    • Synchronize cells at different phases

    • Perform immunoblotting and immunofluorescence

    • Correlate with telomere cohesion timing

  • Investigate potential mitotic functions:

    • Spindle association

    • Chromosome segregation

    • Nuclear envelope reassembly

• Interaction with other chromosome 19 genes:

  • Explore functional relationships with tumor suppressors:

    • SIRT6 (histone deacetylase)

    • BBC3/PUMA (p53 upregulated modulator of apoptosis)

    • STK11 (serine/threonine kinase 11)

    • CADM4 (cell adhesion molecule 4)

    • GLTSCR2 (glioma tumor suppressor candidate region gene 2)

Methodological approach: For these emerging applications, researchers should employ antibodies validated for specific techniques (IP, IF, IHC) and consider developing conditional knockout systems to study tissue-specific functions.

What are the current technical limitations of C19orf43 antibodies and how might they be addressed through methodological innovations?

Current C19orf43 antibody limitations and potential solutions:

Technical limitations and innovations:

• Epitope accessibility challenges:

  • Current limitation: C19orf43's interactions with tankyrase or RNA may mask epitopes

  • Innovative approach:

    • Develop antibodies against multiple epitopes distributed across the protein

    • Create conformation-specific antibodies that recognize distinct structural states

    • Engineer smaller antibody fragments (Fabs, nanobodies) for better accessibility

• Post-translational modification detection:

  • Current limitation: No phospho-specific or other PTM-specific antibodies available

  • Innovative approach:

    • Develop phospho-specific antibodies targeting potential regulatory sites

    • Create antibodies specific to other modifications (acetylation, ubiquitination)

    • Apply mass spectrometry to identify modification sites for targeted antibody development

• Cross-reactivity concerns:

  • Current limitation: Potential cross-reactivity with related proteins in complex samples

  • Innovative approach:

    • Employ CRISPR-engineered knockout cells for validation

    • Use competitive peptide blocking to demonstrate specificity

    • Apply new negative-selection approaches during antibody development

• Quantification limitations:

  • Current limitation: Semi-quantitative nature of many antibody-based techniques

  • Innovative approach:

    • Develop calibrated immunoassays with recombinant protein standards

    • Create bi-epitopic sandwich ELISA systems for improved specificity and sensitivity

    • Implement advanced image analysis for quantitative immunofluorescence

• Functional blockade capability:

  • Current limitation: Most antibodies detect but don't functionally inhibit C19orf43

  • Innovative approach:

    • Screen for antibodies that block exoribonuclease activity

    • Develop antibodies targeting the tankyrase-binding motifs to disrupt interactions

    • Engineer intrabodies that can modulate function in living cells

Methodological recommendation: Researchers should consider employing multiparametric approaches that combine antibody-based detection with orthogonal techniques (mass spectrometry, genetic manipulation, activity assays) to overcome current limitations.