ZRSR2 Antibody

What is ZRSR2 and what is its role in cellular function?

ZRSR2 is a spliceosomal protein that plays a pivotal role in the splicing of U12-type introns, while having minimal effect on U2-dependent splicing. It contains a CCCH-type zinc finger domain, an RNA-binding motif, and serine/arginine-rich regions that are characteristic of splicing factors . ZRSR2 interacts with several splicing machinery components, including members of the SF3B complex, U2AF1, U2AF2, and SRPK1, forming a functional network essential for proper pre-mRNA processing . Loss of ZRSR2 function results in impaired splicing of U12-type introns, which represent a minor but functionally significant subset of introns in the human genome. This splicing defect can lead to altered gene expression patterns that affect cellular differentiation and proliferation, particularly in hematopoietic cells .

How can I distinguish between specific and non-specific binding when using ZRSR2 antibodies?

To ensure specific binding when using ZRSR2 antibodies, employ multiple validation approaches:

• Use appropriate negative controls, including isotype controls and samples where ZRSR2 is knocked down via shRNA (as demonstrated in studies where ZRSR2 sh1 and sh2 vectors resulted in efficient downregulation) .

• Confirm specificity by testing the antibody against recombinant ZRSR2 protein or ZRSR2-overexpressing cells alongside wild-type cells.

• Perform competition assays using the immunizing peptide to block specific binding sites.

• When conducting Western blotting, verify that the detected band aligns with the expected molecular weight of ZRSR2 (~482 amino acids) .

• Cross-validate results using multiple antibodies targeting different epitopes of ZRSR2, such as those directed against the C-terminal region (AA 453-482) versus mid-region epitopes (AA 191-372) .

What applications are ZRSR2 antibodies suitable for in research settings?

ZRSR2 antibodies are suitable for various research applications based on their validated reactivity profiles:

Different antibodies show varying cross-reactivity profiles, with some reacting against human ZRSR2 exclusively, while others recognize homologs across multiple species including mouse, rat, horse, dog, cow, rabbit, guinea pig, and monkey .

How do ZRSR2 mutations affect RNA splicing patterns, and how can ZRSR2 antibodies help characterize these effects?

ZRSR2 mutations in myelodysplastic syndromes (MDS) cause specific splicing aberrations predominantly affecting U12-type introns. These mutations are typically inactivating alterations (nonsense, frame-shift, and splice site mutations) that result in loss of ZRSR2 function .

The splicing defects can be characterized using ZRSR2 antibodies through:

• Differential splicing analysis: Compare splicing patterns between ZRSR2 mutant and wild-type samples using RT-PCR and RNA-seq with immunoprecipitation of ZRSR2-associated transcripts. Analysis of MDS bone marrow samples with ZRSR2 mutations revealed 689 mis-spliced junctions, with a striking overabundance of retained U12-type introns .

• Splicing complex composition studies: Immunoprecipitation with ZRSR2 antibodies coupled with mass spectrometry identified key ZRSR2 interacting partners including SF3B complex members, U2AF1, U2AF2, and SRPK1, as well as novel partners such as C1QBP and CCDC97 .

• Functional validation: ZRSR2 antibodies can validate knockdown efficiency in experimental models designed to recapitulate MDS splicing phenotypes. For example, lentiviral shRNA-mediated knockdown of ZRSR2 resulted in aberrant retention of U12-type introns similar to that observed in patient samples .

The specificity of ZRSR2's role in U12-type intron splicing is remarkable—in comparisons between ZRSR2 mutant and wild-type samples, 43 out of 45 introns consistently retained across all 64 comparison pairs were U12-dependent .

What methods are optimal for detecting ZRSR2-associated R-loops and DNA damage using antibodies?

ZRSR2 loss has been associated with increased R-loop formation and DNA damage. To detect these phenomena:

• R-loop detection:

  • Immunoprecipitate genomic DNA using the S9.6 antibody, which specifically recognizes RNA/DNA hybrids

  • Compare R-loop levels between ZRSR2 knockdown and control cells

  • Normalize to input DNA and quantify by qPCR or sequencing

• DNA damage assessment:

  • Use γH2AX antibodies to detect phosphorylated histone H2AX, a marker of DNA double-strand breaks

  • Perform immunofluorescence microscopy to visualize nuclear foci in ZRSR2-deficient versus control cells

  • Quantify the number and intensity of γH2AX foci

Research has shown a small increase in γH2AX signals in ZRSR2-knockdown cells compared to control cells, suggesting that ZRSR2 deficiency promotes genomic instability . This methodology has helped establish a link between splicing defects and the DNA damage response pathway in MDS pathogenesis.

How can ZRSR2 antibodies be used to study inflammatory signaling in hematopoietic cells with ZRSR2 deficiency?

ZRSR2 deficiency appears to sensitize cells to inflammatory stimuli, particularly through the NF-κB pathway. To investigate this connection using ZRSR2 antibodies:

• Pathway activation analysis: Treat ZRSR2 wild-type and knockdown cells with inflammatory stimuli (e.g., TNFα or LPS), then measure phosphorylated p65 (p-p65) levels using specific antibodies via Western blotting. Research has shown increased p-p65 levels in ZRSR2 knockdown cells following TNFα treatment, indicating enhanced NF-κB activation .

• Protein-protein interaction studies: Use co-immunoprecipitation with ZRSR2 antibodies to identify interactions with components of inflammatory signaling pathways, potentially revealing direct molecular connections.

• ChIP-seq analysis: Combine ZRSR2 antibodies with chromatin immunoprecipitation sequencing to map ZRSR2 binding sites at genes involved in inflammatory responses, potentially uncovering regulatory roles.

• Transcriptional profiling: Compare inflammatory gene expression patterns between ZRSR2-deficient and normal cells using RNA-seq after cytokine stimulation, using ZRSR2 antibodies to validate knockdown efficiency.

This research direction is particularly relevant as activation of innate immune signaling has been proposed as a unifying mechanism mediating the effects of splicing factor mutations in MDS .

What optimization strategies are necessary when using ZRSR2 antibodies for different experimental techniques?

Optimizing ZRSR2 antibody usage requires technique-specific adjustments:

For Western Blotting:

• Antibody dilution: Begin with 1:500-1:1000 for polyclonal antibodies

• Blocking buffer: 5% non-fat milk or BSA in TBST

• Incubation time: Overnight at 4°C for primary antibody

• Detection method: Use enhanced chemiluminescence systems with appropriate sensitivity for low-abundance proteins like ZRSR2

• Sample preparation: Include phosphatase inhibitors if studying phosphorylated forms

For Immunohistochemistry:

• Antigen retrieval: Test both heat-mediated (citrate buffer) and enzymatic methods

• Antibody dilution: Start with 1:100-1:200

• Detection system: Use appropriate amplification systems (e.g., ABC, polymer-based)

• Controls: Include both positive controls (tissues known to express ZRSR2) and negative controls

For Immunoprecipitation:

• Pre-clearing samples to reduce non-specific binding

• Cross-linking conditions when studying protein-RNA interactions

• Buffer composition: Include RNase inhibitors when studying ZRSR2-RNA complexes

• Elution conditions: Optimize to maintain protein activity

For all techniques:

• Validate antibody specificity using ZRSR2 knockdown samples, as demonstrated in studies where lentiviral shRNA vectors efficiently downregulated ZRSR2 transcript and protein levels in 293T cells and leukemia cell lines (TF-1 and K562) .

How can I design experiments to investigate ZRSR2's role in hematopoiesis using appropriate antibodies?

To investigate ZRSR2's role in hematopoiesis:

• Establish knockdown/knockout models:

  • Design shRNA or CRISPR-Cas9 systems targeting ZRSR2

  • Validate knockdown/knockout efficiency by Western blotting with ZRSR2 antibodies

  • Studies have shown that shRNA-mediated knockdown of ZRSR2 inhibits cell growth and alters the in vitro differentiation potential of hematopoietic cells

• Differentiation assays:

  • Culture hematopoietic stem/progenitor cells under conditions promoting differentiation

  • Monitor lineage markers over time by flow cytometry

  • Use ZRSR2 antibodies to correlate expression levels with differentiation stages

• Cell proliferation and colony formation:

  • Compare colony-forming unit (CFU) capacity between ZRSR2-deficient and control cells

  • Measure proliferation using Ki-67 staining or BrdU incorporation

• Intracellular signaling analysis:

  • Use phospho-specific antibodies to detect activation of key hematopoietic signaling pathways in ZRSR2-deficient cells

  • Research has identified increased sensitivity to MAPK pathway inhibitors in ZRSR2 knockdown cells, suggesting altered signaling dynamics

• In vivo models:

  • Transplant ZRSR2-deficient hematopoietic stem cells into irradiated recipients

  • Monitor engraftment, differentiation, and disease development

  • Use ZRSR2 antibodies for immunohistochemical analysis of bone marrow samples

What controls and validation steps are essential when interpreting ZRSR2 antibody data in splicing research?

When using ZRSR2 antibodies in splicing research, implement these controls and validation steps:

• Antibody specificity controls:

  • Include ZRSR2 knockdown or knockout samples as negative controls

  • Test multiple antibodies targeting different epitopes of ZRSR2

  • Perform peptide competition assays to confirm specificity

• Splicing functional controls:

  • Include known U12-type intron-containing transcripts as positive controls

  • Compare results with U2-type intron splicing as a specificity control

  • Studies have consistently shown that ZRSR2 deficiency predominantly affects U12-type introns while U2-dependent splicing remains largely unaffected

• Technical validation:

  • Confirm RNA-seq findings with RT-PCR for selected targets

  • Use multiple primer pairs spanning different exon-intron junctions

  • Validate protein-level consequences of splicing changes by Western blotting

• Biological replicates:

  • Include sufficient biological replicates (≥3) to account for variability

  • For patient samples, stratify by ZRSR2 mutation status and clinical parameters

  • In the published research, eight ZRSR2 mutant MDS samples were compared with ZRSR2 wild-type MDS or normal bone marrow samples using rigorous statistical criteria (FDR<0.01, ΔMSI>20)

• Quantification methods:

  • Use appropriate metrics for splicing efficiency (e.g., Mis-splicing Index)

  • Apply statistical thresholds (e.g., FDR<0.01) for identifying significant splicing events

  • Categorize introns as U12- or U2-type based on established sequence criteria

How can synthetic lethality approaches with ZRSR2 deficiency be leveraged using antibody-based techniques?

Synthetic lethality screens have identified potential therapeutic vulnerabilities in ZRSR2-deficient cells. To leverage these findings using antibody-based techniques:

• Target validation in ZRSR2-deficient cells:

  • Western blotting with antibodies against potential synthetic lethal targets (e.g., ERBB3/4)

  • Immunoprecipitation to detect altered protein interactions in ZRSR2-deficient contexts

  • Screening results have identified increased sensitivity to ERBB3/4 knockdown and MAPK pathway inhibitors in ZRSR2-deficient cells

• Pathway activation monitoring:

  • Use phospho-specific antibodies to monitor activation states of synthetic lethal pathways

  • Measure dynamic changes in response to inhibitor treatment

  • Compare pathway dynamics between ZRSR2 wild-type and deficient cells

• Combinatorial treatment response assessment:

  • Detect markers of apoptosis, cell cycle arrest, or differentiation using specific antibodies following combination treatments

  • Monitor compensatory pathway activation that might indicate resistance mechanisms

• In situ proximity ligation assays:

  • Detect protein-protein interactions between ZRSR2 (or its interacting partners) and components of synthetic lethal pathways

  • Visualize spatial relationships between proteins in intact cells

A systematic approach using these techniques can help translate initial screening findings into mechanistic understanding and therapeutic strategies for MDS patients with ZRSR2 mutations.

What are the key considerations when using ZRSR2 antibodies to analyze patient samples for diagnostic or research purposes?

When analyzing patient samples with ZRSR2 antibodies, consider:

• Sample preparation optimization:

  • For bone marrow biopsies, optimize fixation and antigen retrieval protocols

  • For peripheral blood, develop enrichment strategies for rare cell populations

  • Consider the impact of sample age and storage conditions on epitope integrity

• Mutation-specific considerations:

  • Determine whether the ZRSR2 mutations present affect antibody epitopes

  • Note that ZRSR2 mutations in MDS are often inactivating alterations (nonsense, frame-shift, splice site mutations)

  • Choose antibodies recognizing epitopes upstream of common mutation sites

• Correlative analyses:

  • Compare ZRSR2 expression with clinical parameters (WHO subtype, IPSS score, cytogenetics)

  • Studies have shown ZRSR2 mutations are more prevalent in MDS subtypes without ring sideroblasts and in chronic myelomonocytic leukemia (CMML)

  • Correlate with markers of aberrant splicing or inflammatory activation

• Reference standards:

  • Establish appropriate control samples (age-matched, gender-matched)

  • Note that ZRSR2 mutations primarily affect males due to its X-chromosome location

  • Use standardized scoring systems for immunohistochemical analysis

• Technical validation:

  • Confirm immunohistochemistry findings with orthogonal techniques (e.g., RNA-seq for splicing aberrations)

  • Use digital pathology tools for quantitative assessment of staining patterns

Patient data should include detailed clinical information as shown in the literature, where samples are characterized by age, gender, WHO subtype, IPSS score, and cytogenetic findings .

How can phosphorylation status of ZRSR2 be assessed, and what is its functional significance?

ZRSR2 contains serine/arginine-rich regions that are potential phosphorylation sites regulating its function. To assess ZRSR2 phosphorylation:

• Phosphorylation-specific detection methods:

  • Use phospho-specific antibodies if available

  • Employ Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Perform mass spectrometry analysis of immunoprecipitated ZRSR2 to map phosphorylation sites

• Functional modulation experiments:

  • Treat cells with phosphatase inhibitors to enhance phosphorylation signals

  • Use kinase inhibitors to identify regulatory pathways (particularly SRPK1, which was identified as a ZRSR2 interactor)

  • Create phosphomimetic and phospho-dead mutants of key residues to assess functional consequences

• Context-dependent regulation:

  • Compare phosphorylation patterns during different cell cycle stages

  • Assess changes during hematopoietic differentiation

  • Monitor alterations in response to inflammatory stimuli like TNFα, which has been shown to induce differential responses in ZRSR2-deficient cells

• Correlation with splicing activity:

  • Perform in vitro splicing assays with phosphorylated and dephosphorylated ZRSR2

  • Compare binding affinity to RNA and protein partners under different phosphorylation states

Understanding ZRSR2 phosphorylation may provide insights into its regulation and potential therapeutic approaches targeting its activity in disease contexts.

What emerging technologies can enhance the utility of ZRSR2 antibodies in splicing and hematological research?

Several emerging technologies can expand ZRSR2 antibody applications:

• Single-cell antibody-based technologies:

  • Combine ZRSR2 antibodies with single-cell RNA-seq to correlate protein levels with splicing patterns at single-cell resolution

  • Use mass cytometry (CyTOF) with ZRSR2 antibodies to analyze protein expression across heterogeneous cell populations

  • Implement proximity extension assays for ultrasensitive detection in limited sample material

• Spatial biology approaches:

  • Apply multiplexed immunofluorescence to visualize ZRSR2 alongside other splicing factors in tissue context

  • Implement in situ sequencing to correlate ZRSR2 localization with splicing outcomes in intact tissues

  • Use spatial transcriptomics to map splicing patterns in relation to ZRSR2 expression

• Dynamic interaction monitoring:

  • Employ FRET-based biosensors to track ZRSR2 interactions in living cells

  • Implement optogenetic approaches to modulate ZRSR2 activity with spatial and temporal precision

  • Use live-cell imaging with fluorescently tagged antibody fragments to track ZRSR2 dynamics

• High-throughput functional screening:

  • Develop CRISPR activation/inhibition screens targeting ZRSR2 and interacting partners

  • Implement antibody-based readouts in high-content imaging screens

  • Research has already employed siRNA screens against the tyrosine kinome and small-molecule panels to identify synthetic lethal interactions with ZRSR2 deficiency

These technologies promise to advance our understanding of ZRSR2's role in normal and malignant hematopoiesis.

How can computational approaches be integrated with ZRSR2 antibody data to enhance splicing research?

Integrating computational approaches with ZRSR2 antibody data can yield deeper insights:

• Integrative multi-omics analysis:

  • Correlate ZRSR2 immunoprecipitation data with RNA-seq, CLIP-seq, and proteomics

  • Develop network models incorporating ZRSR2 protein interactions and splicing outcomes

  • Implement machine learning algorithms to predict the impact of ZRSR2 mutations on splicing patterns

• Structural biology integration:

  • Use structural prediction tools to model ZRSR2-antibody binding interfaces

  • Simulate the impact of mutations on ZRSR2 protein structure and function

  • Design epitope-specific antibodies based on structural information

• Splicing outcome prediction:

  • Develop algorithms to predict U12-type intron retention based on ZRSR2 expression levels

  • Create computational pipelines specifically optimized for minor intron detection

  • Studies have shown that introns can be categorized as U2- or U12-type based on the divergence at the 5' and 3' splice sites and the branchpoint sequence

• Clinical data correlation:

  • Implement statistical methods to correlate ZRSR2 protein levels with clinical outcomes

  • Develop predictive models for treatment response based on ZRSR2 status

  • Create decision support tools for precision medicine approaches in MDS