RBM45 Antibody

What is RBM45 and why is it significant in neuroscience research?

RBM45 is an RNA-binding protein that contains RNA recognition motifs with sequence similarities to TDP-43 and FUS proteins, which are implicated in neurodegenerative diseases. It is highly expressed in the developing brain and has been identified as an m6A-binding protein that recognizes methylated RNA via two C-terminal RNA-binding domains (RBDs) . Its significance in neuroscience stems from its role in neuronal differentiation and its association with neurodegenerative conditions including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer's disease (AD) . RBM45 has been detected at altered levels in the cerebrospinal fluid of ALS patients, making it a potential biomarker and therapeutic target for neurological disorders .

What is the subcellular localization pattern of RBM45 in normal tissues?

In normal central nervous system tissues, RBM45 exhibits a predominantly nuclear localization with a distinctive punctate staining pattern within nuclei of both neurons and glial cells in the brain and spinal cord . This nuclear localization is consistent with its function in RNA processing. While RBM45 is predominantly nuclear in healthy cells, it does not exclusively remain there, as it has been observed to shuttle between the nucleus and cytoplasm depending on cellular conditions. Understanding this normal distribution pattern is essential for interpreting pathological changes in disease states .

What are the primary RNA targets of RBM45 and how does it interact with them?

RBM45 binds to thousands of cellular RNAs, primarily within intronic regions. CLIP-seq experiments in mHippoE-2 and HEK293T cells identified 16,399 peaks in 6,495 RNAs and 13,758 peaks in 5,230 RNAs, respectively, with a high degree of target overlap (1,868 RNAs) between mouse and human cells . RBM45 preferentially binds to specific RNA sequences, such as the octanucleotide (GGGACGGU) identified in parvovirus B19 studies . Its binding to certain RNA targets is m6A-dependent, as METTL3 depletion reduced RBM45 binding at 5-15% of identified binding sites . This binding pattern suggests RBM45 plays a key role in pre-mRNA processing, particularly in constitutive splicing mechanisms.

What criteria should be considered when selecting an RBM45 antibody for immunohistochemistry studies?

When selecting an RBM45 antibody for immunohistochemistry (IHC), researchers should consider several critical factors:

• Epitope specificity: Verify that the antibody recognizes specific epitopes of human RBM45 with minimal cross-reactivity to related RNA-binding proteins.

• Validation in relevant tissues: Ensure the antibody has been validated in neural tissues where RBM45 is predominantly expressed.

• Detection of both normal and pathological forms: The ideal antibody should detect both the normal nuclear punctate pattern and pathological cytoplasmic inclusions .

• Compatibility with fixation methods: Confirm the antibody works with your preferred fixation protocol (formalin-fixed paraffin-embedded or frozen sections).

• Performance in co-localization studies: Since RBM45 pathology often involves co-localization with TDP-43 and ubiquitin, ensure the antibody is compatible with multiple labeling techniques .

Available literature demonstrates successful IHC visualization of RBM45 in both normal punctate nuclear distribution and in pathological cytoplasmic inclusions, particularly in ALS, FTLD-TDP, and AD tissues .

How can researchers validate the specificity of an RBM45 antibody?

A comprehensive validation approach for RBM45 antibodies should include:

• Western blot analysis: Verify a single band of appropriate molecular weight (~53 kDa) in tissue extracts or CSF samples, as demonstrated in ALS patient studies .

• Peptide competition assays: Pre-incubation with the immunizing peptide should abolish signal.

• Knockout/knockdown controls: Use CRISPR-Cas9 generated RBM45 knockout cell lines (e.g., mHippoE-2 RBM45 KO) to confirm antibody specificity .

• Multiple antibody comparison: Use antibodies targeting different epitopes of RBM45 to confirm staining patterns.

• Cross-species reactivity assessment: Test antibody performance across relevant species if conducting comparative or animal model studies.

This multi-faceted validation ensures that experimental observations truly reflect RBM45 biology rather than technical artifacts.

How does RBM45 pathology compare across different neurodegenerative diseases?

RBM45 pathology varies across neurodegenerative conditions but shows some consistent patterns:

Unlike TDP-43, which typically shows nuclear clearing in cells with cytoplasmic inclusions, RBM45 maintains some nuclear presence even in cells with cytoplasmic pathology . This distinctive pattern may provide insights into disease mechanisms and progression that differ from those associated with TDP-43 pathology alone.

What methodological approaches can be used to investigate RBM45 pathology in human tissue samples?

Investigating RBM45 pathology in human tissues requires multi-modal approaches:

• Immunohistochemistry on FFPE sections: Using validated RBM45 antibodies with DAB chromogen to visualize distribution patterns.

• Immunofluorescence co-localization studies: Double or triple labeling with antibodies against RBM45, TDP-43, ubiquitin, and other relevant proteins.

• Quantitative assessment: Count and characterize RBM45-positive inclusions across brain regions and cell types.

• Biochemical fractionation: Separate nuclear and cytoplasmic fractions followed by western blotting to assess RBM45 distribution changes .

• Proteomic analysis of CSF: Liquid chromatography tandem mass spectrometry to detect altered RBM45 levels in patient CSF samples .

These complementary approaches can provide a comprehensive picture of RBM45 pathology and its relationship to disease mechanisms and clinical features.

How does RBM45 regulate RNA splicing and what experimental methods can detect these effects?

RBM45 regulates constitutive splicing of target pre-mRNAs through both m6A-dependent and m6A-independent mechanisms. Its depletion disrupts splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels . To investigate these effects, researchers can employ:

• RNA-seq analysis: Compare splicing patterns between RBM45 knockout and control cells to identify differentially spliced transcripts.

• qRT-PCR with intron-spanning primers: Assess splicing efficiency at specific intron-exon boundaries, as demonstrated with the Ide pre-mRNA in mHippoE-2 cells .

• CLIP-seq: Identify direct RBM45 binding sites on target RNAs and correlate with splicing changes .

• Splicing reporter assays: Construct minigene reporters containing suspected RBM45-dependent splicing regions.

• RNA pulldown assays: Identify RNA sequences bound by RBM45, as demonstrated with the octanucleotide (GGGACGGU) in parvovirus B19 studies .

These approaches can elucidate how RBM45 contributes to normal RNA processing and how its dysfunction may contribute to disease states.

What is the relationship between RBM45 and m6A RNA modification?

RBM45 functions as an m6A-binding protein that recognizes methylated RNA sites via its C-terminal RNA-binding domains. The relationship between RBM45 and m6A modification can be characterized as follows:

• Direct binding to m6A sites: RBM45 directly binds m6A-modified RNA through its RNA recognition motifs .

• m6A-dependent binding to subset of targets: Depletion of METTL3 (a key m6A methyltransferase) reduces RBM45 binding to 5-15% of its normal target sites .

• Dual recognition mechanism: RBM45 exhibits both m6A-dependent and m6A-independent RNA binding capabilities .

• Functional consequences: The m6A-dependent binding of RBM45 influences subsequent RNA processing steps, including splicing regulation.

To study this relationship, researchers can use METTL3 knockdown/knockout systems in conjunction with RBM45 binding studies (CLIP-seq) and RNA processing analysis (RNA-seq) to distinguish between m6A-dependent and independent functions .

What are the optimal conditions for immunoprecipitating RBM45 for protein interaction studies?

For successful RBM45 immunoprecipitation (IP), researchers should consider:

• Lysis buffer composition: Use buffers containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, with protease and phosphatase inhibitors.

• Cross-linking approach: For transient interactions, consider formaldehyde cross-linking (0.1-1% for 10 minutes) before cell lysis.

• Nuclear extraction: Since RBM45 is predominantly nuclear, specialized nuclear extraction protocols may improve yield.

• RNase treatment: Include both RNase-treated and untreated samples to distinguish RNA-dependent and independent interactions.

• IP antibody selection: Use antibodies validated for IP applications, potentially with epitope tags (HA-RBM45) for enhanced specificity .

These conditions have successfully identified RBM45 interactions with splicing factors and other RNA-binding proteins in previous studies .

How can researchers establish and validate RBM45 knockout models?

Establishing reliable RBM45 knockout models requires:

• CRISPR-Cas9 system: Design sgRNAs targeting early exons of RBM45 as performed in mHippoE-2 cells .

• Validation strategies:

  • Western blot confirmation of complete protein loss

  • Genomic DNA sequencing of the targeted region

  • qRT-PCR to confirm reduced mRNA levels

• Control lines: Generate both Cas9-only control cells (without sgRNA) and rescue lines expressing RBM45 cDNA to confirm phenotype specificity .

• Phenotypic analysis: Assess:

  • RNA splicing changes via RNA-seq

  • Cell proliferation and differentiation, particularly in neuronal models

  • Expression of RBM45 target genes

Published RBM45 knockout models in mHippoE-2 cells have shown specific gene expression changes affecting 668 RNAs, with 46 high-confidence targets showing both binding and expression changes that were reversible with RBM45 restoration .

How does RBM45 contribute to neuronal differentiation and what methods best capture these effects?

RBM45 plays a crucial role in neuronal differentiation, with its depletion compromising both proliferation and differentiation of human neuroblastoma cells (SH-SY5Y) . To investigate this function:

• Neuroblastoma differentiation models: Use SH-SY5Y cells with retinoic acid/BDNF treatment protocols under RBM45 knockdown or knockout conditions.

• Gene expression profiling: Analyze transcriptome changes during differentiation with and without RBM45.

• Neurodevelopmental pathway analysis: Focus on signaling pathways affected by RBM45 depletion.

• Morphological assessment: Quantify neurite outgrowth, branching complexity, and neuronal marker expression.

• Rescue experiments: Reintroduce wild-type or domain-mutant RBM45 to determine which functions are essential for differentiation.

RBM45 depletion has been shown to dampen gene expression changes that normally occur during neuronal differentiation, an effect that can be rescued by restoring RBM45 expression .

What are the methodological considerations for studying RBM45's role in viral infections?

RBM45 has been identified as a key regulator of viral RNA processing, particularly in parvovirus B19 infection. When investigating these functions:

• Viral infection models: Establish appropriate cell systems (such as CD36+ erythroid progenitor cells for B19V) .

• RNA-protein interaction assays:

  • RNA pulldown with biotinylated viral RNA sequences

  • In vitro binding assays to identify binding sites

  • CLIP-seq in infected cells

• Functional validation:

  • RBM45 knockdown followed by viral RNA splicing analysis

  • Viral DNA replication assessment via Southern blotting

  • Viral protein expression analysis via Western blotting

• Mutagenesis: Introduce silent mutations in viral RNA binding sites (like ISE2 and ISE3 in B19V) to confirm functional relevance .

Studies have shown that RBM45 binds to specific intronic splicing enhancers and is essential for the maturation of viral mRNAs encoding specific proteins like the 11-kDa protein in B19V, affecting viral replication .

How can researchers address common technical challenges when working with RBM45 antibodies?

Several technical challenges may arise when working with RBM45 antibodies:

• Nuclear antigen accessibility: For optimal nuclear staining:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Compare fixation protocols (4% PFA vs. formalin)

  • Consider permeabilization optimization (0.1-0.5% Triton X-100)

• Distinguishing normal vs. pathological RBM45:

  • Use confocal microscopy for improved resolution of nuclear punctate staining

  • Employ Z-stack imaging to fully capture 3D distribution patterns

  • Consider super-resolution techniques for detailed subcellular localization

• Cross-reactivity issues:

  • Include knockout controls whenever possible

  • Test multiple commercial antibodies targeting different epitopes

  • Consider preabsorption with recombinant RBM45 to confirm specificity

• Low signal detection:

  • Try signal amplification methods (TSA, polymer detection systems)

  • Optimize antibody concentration through titration experiments

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

These approaches can help overcome technical limitations and produce more reliable results when studying RBM45 biology.

What are the emerging research questions regarding RBM45 in neurological disease?

Several promising research directions for RBM45 investigation include:

• RBM45 as a biomarker: Further validation of altered CSF levels in neurodegenerative diseases for diagnostic and prognostic applications .

• Relationship to C9ORF72 pathology: Deeper investigation into why RBM45 pathology is most extensive in C9ORF72 mutation carriers .

• Therapeutic targeting: Development of approaches to modulate RBM45 function or prevent its pathological aggregation.

• RNA splicing regulation: Comprehensive mapping of RBM45-dependent splicing events in the brain and their dysregulation in disease.

• Protein interaction networks: Identification of RBM45 protein partners in normal and disease states to understand functional consequences of its mislocalization.

These emerging questions represent critical gaps in our understanding of RBM45 biology and its contributions to neurological disease pathogenesis.

What experimental approaches can distinguish between RBM45's direct effects and secondary consequences?

Distinguishing direct from indirect effects of RBM45 requires sophisticated experimental designs:

• Temporal analysis: Use inducible knockdown/knockout systems to track immediate versus delayed effects.

• Domain mutant expression: Express specific RBM45 domain mutants to dissect which functions are essential for observed phenotypes.

• Direct binding site identification: Combine CLIP-seq with RNA-seq to correlate binding sites with functional outcomes .

• Rescue experiments with specific constructs: Rescue RBM45 knockout with constructs lacking specific domains or containing point mutations in functional regions.

• In vitro reconstitution: Use purified components to test if RBM45 alone is sufficient for observed RNA processing effects.

These approaches can help distinguish which cellular processes are directly regulated by RBM45 versus those that change as secondary consequences of RBM45 manipulation.