PRMT9 Antibody, Biotin conjugated

What is PRMT9 and what is its primary function in cellular processes?

PRMT9 (protein arginine methyltransferase 9) is a type II methyltransferase that generates symmetrically di-methylated arginine (SDMA) on target proteins. It contains two S-adenosylmethionine binding motifs and three tetratricopeptide repeats (TPRs) . PRMT9 plays a critical role in post-translational modifications involved in various cellular functions, including RNA processing, gene expression, genome stability, and signal transduction .

The primary established function of PRMT9 is the methylation of the splicing factor SF3B2 (also known as SAP145) at arginine 508, which modulates pre-mRNA splicing events . This methylation affects SF3B2's interaction with RNA in a context-dependent manner, influencing 3' splice site selection and contributing to alternative splicing regulation .

How does PRMT9 differ from other protein arginine methyltransferases?

PRMT9 is classified as a type II methyltransferase, specifically generating symmetrical dimethylarginine (SDMA) modifications. Unlike some other PRMTs, PRMT9 demonstrates high substrate specificity, with SF3B2 (specifically at arginine 508) being its primary validated methylation target . Recent research strongly indicates that SF3B2 is the principal substrate of PRMT9, highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing .

PRMT9 is also distinguished by its cellular distribution pattern and unique regulatory mechanisms. The enzyme shows both nuclear and cytoplasmic localization, which is maintained even in mutant forms that lack catalytic activity . Additionally, PRMT9 appears to be regulated through ubiquitination processes involving E3 ubiquitin ligase UBE3C and molecular chaperone VCP/p97, which dictate its stability and degradation pathways .

What are the optimal applications for PRMT9 antibodies in experimental research?

PRMT9 antibodies can be effectively employed in multiple research applications, though their optimal use depends on specific experimental objectives. Based on available product information, PRMT9 antibodies have been validated for immunofluorescence (IF) and immunocytochemistry (ICC) applications, making them particularly suitable for subcellular localization studies . These applications allow researchers to visualize PRMT9 distribution within cellular compartments and potentially track changes in its localization under different experimental conditions.

For comprehensive PRMT9 research, antibodies can also be used in:

• Co-immunoprecipitation (co-IP) assays to study protein-protein interactions, particularly between PRMT9 and its substrate SF3B2 or regulatory proteins like UBE3C

• Western blot analysis to detect expression levels and protein stability in various experimental conditions

• Chromatin immunoprecipitation (ChIP) experiments to investigate potential roles in transcriptional regulation

When selecting antibodies for these applications, researchers should consider the epitope recognition properties and validate specificity in their experimental system, as downstream results rely heavily on antibody quality.

How can researchers validate the specificity of PRMT9 antibodies?

Validating antibody specificity is critical for ensuring experimental reliability. For PRMT9 antibodies, a multi-step validation approach is recommended:

• Knockout/knockdown controls: Compare antibody reactivity in wild-type samples versus PRMT9 knockout or knockdown samples. Complete absence of signal in knockout samples strongly supports specificity .

• Overexpression controls: Test antibody reactivity against samples overexpressing tagged PRMT9 constructs, comparing signal intensity with endogenous expression levels.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (when available, such as "a synthesized peptide derived from human PRMT9" ) before application to samples. Signal disappearance indicates specific binding.

• Cross-reactivity assessment: Test against related proteins (other PRMTs) to ensure the antibody doesn't recognize homologous regions in similar proteins.

• Multiple antibody comparison: When possible, compare results using antibodies raised against different PRMT9 epitopes to confirm consistent findings.

For antibodies targeting specific post-translational modifications (such as those detecting SF3B2 R508me2s), additional validation using site-specific mutants (e.g., R508K) is essential to confirm modification-specific detection rather than protein-level recognition .

What techniques can researchers use to study PRMT9 enzymatic activity?

Several methodological approaches can be employed to investigate PRMT9 enzymatic activity:

In vitro methylation assays: These assays directly measure PRMT9's ability to methylate substrates. The PRMT9 Homogeneous Assay Kit provides a convenient 384-well AlphaLISA® format for quantitatively measuring PRMT9 activity . The principle involves:

• Pre-incubation of PRMT9 enzyme with potential inhibitors

• Addition of substrate mixture to initiate the methyltransferase reaction

• Sequential addition of acceptor beads, primary antibody against methylated peptides, and donor beads

• Measurement of Alpha-counts, which are proportional to PRMT9 activity

Site-specific methylation detection: Researchers can utilize antibodies specifically recognizing methylated substrates, such as the αSF3B2 R508me2s antibody that detects symmetrically dimethylated arginine 508 of SF3B2 . This approach allows for monitoring PRMT9 activity in cellular contexts through immunoblotting or immunofluorescence techniques.

Mass spectrometry-based approaches: Liquid chromatography-mass spectrometry (LC/MS) can be used to identify and quantify methylated peptides in PRMT9 substrate proteins, providing precise information about methylation sites and stoichiometry .

What are the methodological considerations for studying PRMT9-mediated protein methylation?

When investigating PRMT9-mediated protein methylation, researchers should consider several methodological factors:

• Appropriate expression systems: Recombinant PRMT9 should be expressed with proper tags (e.g., FLAG-Tag, His-Tag) for purification while maintaining enzymatic activity .

• Substrate preparation: The confirmed substrate SAP145/SF3B2 (amino acids 401-550) should be properly expressed and purified, with appropriate tags (e.g., GST-Tag, Biotin-labeled) for detection or immobilization .

• Cofactor requirements: Ensure adequate S-adenosylmethionine (SAM) availability, as it serves as the methyl donor for the reaction . The recommended concentration is provided in assay kits (typically 100 μM) .

• Buffer optimization: Use optimized buffers such as 4x HMT Assay Buffer 2A for the enzymatic reaction and appropriate detection buffers for assay readout .

• Temperature and incubation time: These parameters should be optimized based on the specific assay format and PRMT9 activity level.

• Controls inclusion: Always include both positive controls (wild-type PRMT9 with substrate) and negative controls (catalytically inactive PRMT9 mutants, such as the VLDI to AAAA "4A" mutant) .

• Storage considerations: Maintain enzyme and reagent stability by following proper storage recommendations (typically -80°C for PRMT9 enzyme and substrate preparations) .

What is the evidence for PRMT9's role in neurological development and disorders?

Recent research has established significant connections between PRMT9 dysfunction and neurological development and disorders:

• Autosomal Recessive Intellectual Disability (ARID): Loss-of-function mutations in PRMT9, such as the G189R mutation, have been implicated in ARID . This mutation completely abolishes PRMT9's catalytic activity on SF3B2 R508 methylation and leads to heavy PRMT9 ubiquitination .

• Synapse development: Conditional knockout (cKO) of Prmt9 in excitatory neurons leads to aberrant synapse development in mouse models, establishing a causal relationship between PRMT9 loss-of-function and developmental abnormalities .

• Learning and memory: Prmt9 cKO mice exhibit impaired learning and memory, further supporting PRMT9's crucial role in cognitive function development .

• Mechanistic pathway: The neuronal defects observed in Prmt9 cKO mice are likely attributable to SF3B2 methylation deficiency, as similar phenotypes are observed in SF3B2 methylation-deficient knock-in (KI) mouse models . This suggests the PRMT9/SF3B2 axis as a critical regulatory pathway in neuronal development.

• Splicing regulation: Mechanistically, R508 methylation modulates SF3B2–anchoring site interaction in a context-dependent manner, influencing 3' splice site selection and potentially affecting the splicing of neurodevelopmentally important genes .

How is PRMT9 activity implicated in cancer biology?

PRMT9 has shown increased activity in specific cancer contexts, particularly in hematological malignancies:

• Acute Myeloid Leukemia (AML): PRMT9 exhibits increased activity in blast and leukemia stem cells (LSCs) derived from patients with AML, suggesting a potential role in cancer cell survival and proliferation .

• DNA damage response: PRMT9 inhibition increases DNA damage in cancer cells, potentially disrupting their ability to repair genomic lesions and maintain chromosomal stability .

• Immune evasion: PRMT9 activity appears to modulate type I interferon (IFN) responses, with inhibition enhancing these responses . This suggests PRMT9 may contribute to immune evasion mechanisms in cancer cells.

• Therapeutic implications: The relationship between PRMT9 and immune response pathways suggests potential for combination therapy approaches. For instance, combining PD-1/PD-L1 inhibitors with PRMT9 inhibitors may provide synergistic benefits in oncology treatments .

• Cellular stability regulation: The UBE3C/PRMT9 axis identified in recent research may represent a novel regulatory mechanism for protein stability that could be dysregulated in cancer contexts .

How do mutations affect PRMT9 function and stability?

The G189R mutation, identified in individuals with autosomal recessive intellectual disability, provides significant insights into how mutations can affect PRMT9 function and stability:

• Catalytic activity loss: The G189R mutation completely abolishes PRMT9's methyltransferase activity, preventing it from methylating SF3B2, its primary substrate . This loss of function was demonstrated through in vitro methylation assays comparing wild-type, G189R mutant, and the established catalytic-inactive 4A mutant .

• Substrate interaction impairment: G189R mutant PRMT9 fails to interact with SF3B2 in co-immunoprecipitation experiments, indicating that the mutation disrupts critical protein-protein interactions necessary for substrate recognition .

• Protein stability reduction: The G189R mutation leads to severe protein instability. Specifically, G189R-mutant PRMT9 is heavily ubiquitinated in cells, resulting in accelerated proteasomal degradation . This ubiquitination is so extensive that it produces a non-resolvable smear of poly-ubiquitinated protein in gel electrophoresis .

• E3 ligase interaction enhancement: The mutant form shows stronger interaction with UBE3C (an E3 ubiquitin ligase) and VCP/p97 (a molecular chaperone that guides ubiquitinated proteins to proteasomes), compared to wild-type PRMT9 . This enhanced interaction likely explains the increased ubiquitination and reduced stability.

• Protein half-life reduction: The half-life of G189R-mutant PRMT9 is drastically reduced to 1-2 hours compared to wild-type protein, though this can be extended to over 8 hours when UBE3C is knocked down .

What experimental approaches can researchers use to investigate PRMT9's role in alternative splicing?

Researchers can employ several sophisticated experimental approaches to investigate PRMT9's role in alternative splicing:

• RNA-seq analysis in PRMT9-deficient models: Comparing transcriptome-wide splicing patterns between wild-type and PRMT9 knockout or knockdown models can identify specific splicing events regulated by PRMT9. This can be complemented with rescue experiments using wild-type versus catalytically inactive PRMT9 to establish methylation-dependent effects .

• SF3B2 methylation-specific studies: Utilizing SF3B2 methylation-deficient models (R508K mutation) allows researchers to distinguish between methylation-dependent and methylation-independent functions of the PRMT9/SF3B2 axis .

• Mechanistic RNA-protein interaction studies: Investigating how R508 methylation affects SF3B2 interaction with pre-mRNA elements, particularly at the anchoring site critical for 3' splice site selection . This can be accomplished through:

  • RNA immunoprecipitation (RIP) assays comparing wild-type and methylation-deficient SF3B2

  • In vitro RNA binding assays with synthesized RNA containing anchoring site sequences

  • Cross-linking immunoprecipitation (CLIP) to map genome-wide SF3B2-RNA interactions

• Proteomic analyses: Mass spectrometry-based approaches to identify differential protein interactions of methylated versus unmethylated SF3B2, providing insights into how PRMT9-mediated methylation might affect spliceosome assembly or function .

• Minigene splicing assays: Constructing reporter systems with specific exon-intron arrangements to directly test how PRMT9 activity affects splicing outcomes for particular genes of interest.

What are the optimal storage and handling conditions for PRMT9 antibodies and related reagents?

Proper storage and handling of PRMT9 antibodies and related reagents are crucial for maintaining their functionality and ensuring experimental reproducibility:

• PRMT9 antibodies: Commercial PRMT9 antibodies should typically be stored according to manufacturer recommendations. For the rabbit polyclonal antibody documented in the search results, appropriate long-term storage conditions should be followed to maintain specificity and sensitivity .

• Recombinant PRMT9 protein: PRMT9 protein preparations (FLAG-Tag, His-Tag) should be stored at -80°C to preserve enzymatic activity . Repeated freeze-thaw cycles should be avoided by aliquoting the protein into single-use volumes.

• Substrate proteins: Purified SAP145/SF3B2 substrate preparations (GST-Tag, Biotin-Labeled) require -80°C storage to maintain structural integrity and prevent degradation .

• S-adenosylmethionine (SAM): As the methyl donor cofactor, SAM is particularly sensitive to degradation. Store 100 μM SAM solutions at -80°C in small aliquots to prevent repeated thawing .

• Assay buffers: Different components require different storage conditions. While 4x HMT Assay Buffer 2A can be stored at -20°C, the 4x Detection Buffer 3 requires -80°C storage to maintain optimal performance .

• Detection reagents: Specialized antibodies for detecting methylated products (such as Primary Antibody 28) should be stored at -80°C in small working aliquots .

• Working with AlphaLISA reagents: When using PRMT9 assay kits with AlphaLISA® detection systems, special handling considerations include:

  • Protecting AlphaLISA® Anti-Rabbit IgG Acceptor Beads and AlphaScreen® Streptavidin-Conjugated Donor Beads from light exposure

  • Allowing reagents to equilibrate to room temperature before opening to prevent condensation

  • Using low-retention tips and tubes for accurate dispensing

What are the common technical challenges when working with PRMT9 antibodies and how can they be overcome?

Researchers working with PRMT9 antibodies may encounter several technical challenges that can be addressed through methodological refinements:

• Specificity concerns:

  • Challenge: Cross-reactivity with other PRMT family members due to conserved domains.

  • Solution: Validate antibody specificity using PRMT9 knockout/knockdown controls and peptide competition assays . Consider using antibodies raised against less conserved regions of PRMT9.

• Variable expression levels:

  • Challenge: PRMT9 expression may vary across tissues and cell types, affecting detection sensitivity.

  • Solution: Optimize protein loading amounts for Western blots; for immunofluorescence, adjust exposure settings and use signal amplification techniques when necessary.

• Protein stability issues:

  • Challenge: PRMT9 is subject to ubiquitination and degradation, particularly certain mutant forms .

  • Solution: Include proteasome inhibitors (e.g., MG132) in lysis buffers when studying unstable variants; process samples rapidly at cold temperatures to minimize degradation.

• Detection of methylated substrates:

  • Challenge: Distinguishing between unmethylated and methylated forms of SF3B2.

  • Solution: Use highly specific antibodies against the symmetrically dimethylated R508 of SF3B2 (αSF3B2 R508me2s) ; include appropriate positive and negative controls in each experiment.

• Subcellular localization variability:

  • Challenge: PRMT9 exhibits both nuclear and cytoplasmic localization, which may change under different conditions.

  • Solution: For immunofluorescence applications, optimize fixation methods (paraformaldehyde vs. methanol) and permeabilization conditions to preserve both nuclear and cytoplasmic signals.

• Batch-to-batch variation:

  • Challenge: Commercial antibodies may show lot-to-lot variability affecting experimental reproducibility.

  • Solution: Validate each new antibody lot against previous lots; maintain reference samples for comparison; document lot numbers used in experiments.