RNMT Antibody

What is RNMT and why is it significant in cellular biology?

RNMT (RNA guanine-7 methyltransferase) is a 476 amino acid nuclear protein with a molecular weight of approximately 55 kDa that functions as the catalytic subunit of the mRNA-capping methyltransferase RNMT:RAMAC complex. This enzyme methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs, which is essential for mRNA processing and translation initiation . RNMT consists of a catalytic domain (residues 121-476) with homology to other eukaryotic cap methyltransferases and an N-terminal regulatory domain (residues 1-120) that mediates recruitment to transcription initiation sites . The methyl cap is crucial for eukaryotic gene expression as it mediates multiple aspects of RNA metabolism including RNA processing, nuclear export, and translation initiation. Recent studies have highlighted RNMT's role in coordinating mRNA, snoRNA, and rRNA production required for ribosome biogenesis, particularly during T cell activation .

What are the structural and functional domains of RNMT?

RNMT consists of two major domains:

• N-terminal domain (amino acids 1-120): This domain is conserved in mammals but not required for cap methyltransferase activity. Research has shown that this domain is necessary and sufficient for RNMT recruitment to transcription initiation sites in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner . The N-terminal domain is required for transcript expression, translation, and cell proliferation, though it's not essential for the catalytic activity itself.

• Catalytic domain (amino acids 121-476): This domain contains the methyltransferase activity responsible for adding the methyl group to the cap structure. It has homology to other eukaryotic cap methyltransferases .

RNMT functions in complex with RAM (RNMT-activating miniprotein), which enhances its catalytic activity. Biophysical and biochemical studies have shown that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT, thereby allosterically activating RNMT .

What species reactivity considerations are important when selecting an RNMT antibody?

When selecting an RNMT antibody, considering species reactivity is crucial for experimental success. Based on available commercial antibodies, the following reactivity profiles should be considered:

RNMT gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species , so cross-reactivity should be considered when working with non-human models. When selecting an antibody for cross-species applications, it's advisable to validate the antibody in your specific model system before conducting extensive experiments.

What are the optimal applications for different RNMT antibody formats?

RNMT antibodies are employed in various experimental applications, with different formats optimized for specific techniques:

For Western blot, RNMT typically appears at approximately 55-60 kDa. Multiple commercial antibodies have been validated for this application, and it's the most widely used technique for RNMT detection . For immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is often recommended, though citrate buffer pH 6.0 may also be used . When designing ChIP experiments to study RNMT recruitment to transcription sites, researchers have successfully used epitope-tagged RNMT (HA-RNMT) in combination with the appropriate antibody-conjugated agarose .

How should I optimize Western blot protocols for RNMT detection?

Optimizing Western blot protocols for RNMT detection requires attention to several key parameters:

• Sample preparation: Nuclear enrichment may improve detection since RNMT is primarily localized in the nucleus.

• Antibody selection and dilution: Commercial RNMT antibodies have varying recommended dilutions:

  • Proteintech 67673-1-Ig: 1:2000-1:10000

  • Proteintech 13743-1-AP: 1:500-1:1000

  • Cell Signaling #57407: 1:1000

• Positive controls: Several cell lines have been validated for RNMT expression and can serve as positive controls:

  • HeLa cells

  • HEK-293 cells

  • Jurkat cells

  • K-562 cells

  • LNCaP cells

  • HepG2 cells

• Detection method: Standard ECL detection is typically sufficient for RNMT visualization.

• Expected molecular weight: RNMT should be detected at approximately 55-60 kDa. The calculated molecular weight is 55 kDa based on its 476 amino acid sequence .

When troubleshooting, consider that high background may require further optimization of blocking conditions or antibody dilution. Non-specific bands may appear, particularly with polyclonal antibodies, so molecular weight markers are essential for correct band identification.

What controls should I include when using RNMT antibodies in immunoprecipitation experiments?

For rigorous immunoprecipitation experiments using RNMT antibodies, the following controls are essential:

• Input control: Reserve a small portion (5-10%) of the pre-cleared lysate to confirm the presence of RNMT in your starting material.

• Negative control immunoprecipitation: Use an isotype-matched irrelevant antibody (for monoclonal) or pre-immune serum (for polyclonal) to assess non-specific binding.

• Positive control immunoprecipitation: If studying RNMT complexes, include a known interacting partner such as RAM as a positive control for co-immunoprecipitation.

• Validation in RNMT-depleted samples: For definitive specificity testing, include samples where RNMT has been knocked down or knocked out using RNA interference or CRISPR-Cas9 technologies.

• Epitope competition: Where applicable, pre-incubation with the immunizing peptide can demonstrate antibody specificity.

For studying the RNMT-RAM complex, researchers have successfully used reciprocal co-immunoprecipitation approaches. For example, in one study, HEK-293 cells were transfected with plasmids encoding HA-RNMT and FLAG-RAM, followed by immunoprecipitation using anti-HA or anti-FLAG antibody-conjugated agarose .

How can RNMT antibodies be used to study mRNA cap methylation dynamics?

RNMT antibodies can be employed in several sophisticated approaches to study mRNA cap methylation dynamics:

• ChIP-seq analysis: RNMT antibodies can be used in chromatin immunoprecipitation followed by sequencing to map RNMT recruitment to transcription start sites genome-wide. This approach has revealed that the N-terminal domain of RNMT is necessary and sufficient for recruitment to transcription initiation sites .

• RNMT-RAM complex dynamics: Using antibodies against both RNMT and RAM in co-immunoprecipitation experiments can reveal how the complex forms and functions in different cellular contexts. The molecular basis of RAM activation of RNMT has been elucidated using such approaches, showing that RAM increases the recruitment of the methyl donor, AdoMet, to RNMT .

• Cell-type specific regulation: Antibodies against RNMT have revealed its upregulation during T cell receptor (TCR) stimulation, where it coordinates mRNA, snoRNA, and rRNA production required for ribosome biogenesis . This application requires careful consideration of antibody specificity in immune cell types.

• Post-translational modification analysis: Immunoprecipitation with RNMT antibodies followed by mass spectrometry can identify post-translational modifications that regulate RNMT activity.

• Temporal dynamics: Using RNMT antibodies in time-course experiments can reveal how cap methylation changes during cellular processes like differentiation or stress responses.

For these approaches, it's critical to validate antibody specificity and optimize experimental conditions for each specific application.

What experimental approaches can be used to investigate RNMT:RAM complex formation and regulation?

Several experimental approaches utilizing RNMT antibodies can elucidate the formation and regulation of the RNMT:RAM complex:

• Co-immunoprecipitation assays: RNMT antibodies can be used to pull down the complex, followed by detection of RAM by Western blot. This approach has been used to demonstrate that RAM is recruited to transcription initiation sites via an interaction with RNMT .

• Domain-specific studies: Antibodies recognizing different domains of RNMT can help determine which regions are essential for RAM interaction. Research has shown that the catalytic domain of RNMT (amino acids 121-476) is sufficient for RAM binding .

• In vitro methyltransferase assays: Purified components of the RNMT:RAM complex can be assayed for activity using methyl donor substrates. Studies have shown that RAM increases the recruitment of AdoMet to RNMT, thereby allosterically activating the enzyme .

• Structural studies: While not directly using antibodies, information from antibody epitope mapping can guide the design of constructs for structural studies of the RNMT:RAM complex.

• Cell-based assays: Antibodies against RNMT and RAM can be used to monitor complex formation under different cellular conditions, such as during cell cycle progression or in response to signaling pathways.

For investigating the allosteric activation of RNMT by RAM, researchers have used a combination of biophysical and biochemical approaches, showing that RAM increases the recruitment of AdoMet to RNMT .

How can I use RNMT antibodies to study its role in T cell activation and ribosome biogenesis?

RNMT antibodies can be powerful tools for investigating its role in T cell activation and ribosome biogenesis:

• Expression analysis during T cell activation: Western blot analysis using RNMT antibodies has shown that RNMT is induced by T cell receptor (TCR) stimulation . Time-course experiments can reveal the kinetics of RNMT upregulation during activation.

• Ribosome biogenesis pathway analysis: RNMT has been shown to coordinate mRNA, snoRNA, and rRNA production required for ribosome biogenesis . Immunoprecipitation of RNMT followed by RNA sequencing can identify the specific RNA targets regulated by RNMT in T cells.

• Regulation of TOP mRNAs: Transcriptomic and proteomic analyses have demonstrated that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, which are targets of the m7G-cap binding protein LARP1 . RNMT antibodies can be used in RNA immunoprecipitation experiments to study this selective regulation.

• Conditional knockout models: In Rnmt conditional knockout CD4 T cells, the expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised, resulting in decreased ribosome synthesis, reduced translation rates, and proliferation failure . RNMT antibodies are essential for validating knockout efficiency in such models.

• Molecular mechanism studies: RNMT antibodies can be used in ChIP experiments to investigate how RNMT is recruited to specific genes involved in ribosome biogenesis during T cell activation.

Recent research has demonstrated that by enhancing ribosome abundance, upregulation of RNMT coordinates mRNA capping and processing with increased translational capacity during T cell activation .

What are the considerations for using mRNA-encoded antibody approaches to deliver RNMT-targeting antibodies?

Recent advances in mRNA-encoded antibody technology offer new possibilities for targeting RNMT:

• Delivery formats: In vitro transcribed (IVT) mRNA encoding therapeutic antibodies can be delivered in conventional immunoglobulin (IgG) format or as engineered scFv intrabodies . For RNMT targeting, both formats have distinct advantages:

  • IgG format: Suitable for targeting extracellular proteins

  • scFv intrabody format: Can engage intracellular RNMT

• mRNA design considerations: For optimal expression of RNMT-targeting antibodies, mRNA should include:

  • 5' UTR with optimal translation efficiency

  • Kozak sequence for efficient translation initiation

  • Appropriate signal peptide (e.g., interleukin-2 secretory peptide for secreted antibodies)

  • 5' cap with Anti-Reverse Cap Analog

  • 3' poly(A) tail (≥150 nucleotides)

• Validation approaches: To confirm successful mRNA-encoded antibody expression and target engagement:

  • Western blot to detect antibody expression

  • Co-localization studies using immunofluorescence

  • Functional assays to assess target engagement

• Delivery challenges: For neurological applications targeting RNMT, crossing the blood-brain barrier remains a significant challenge. Current approaches rely on direct intracranial injection, limiting clinical translation .

• Sequence optimization: High GC content can improve protein expression by helping synthetic mRNA evade degradation and minimize immunogenicity through decreased interaction with Toll-like receptors .

This approach has been successfully demonstrated with the tau-specific antibody RNJ1, where transfection of neuroblastoma cells with mRNA encoding the light chain and heavy chain resulted in expression and successful generation of secreted RNJ1 IgG capable of binding to human tau .

Why might I observe multiple bands when using RNMT antibodies in Western blot?

Multiple bands in RNMT Western blots can occur for several reasons, each requiring specific troubleshooting approaches:

• Isoform detection: Up to 2 different isoforms have been reported for RNMT . The canonical isoform has 476 amino acids with a molecular weight of approximately 55 kDa. If your antibody recognizes an epitope present in multiple isoforms, you may observe additional bands.

• Post-translational modifications: RNMT can undergo modifications that alter its electrophoretic mobility, resulting in multiple bands. These may include phosphorylation, ubiquitination, or other modifications that regulate RNMT activity.

• Proteolytic degradation: Sample preparation without adequate protease inhibitors can lead to partial degradation of RNMT, resulting in lower molecular weight bands. Always use fresh protease inhibitors and keep samples cold during preparation.

• Non-specific binding: Some antibodies, particularly polyclonal ones, may cross-react with proteins sharing similar epitopes. To determine which band represents RNMT:

  • Compare with positive control samples (e.g., recombinant RNMT)

  • Use RNMT knockdown/knockout samples as negative controls

  • Try alternative antibodies recognizing different RNMT epitopes

• Antibody contamination: Secondary antibody contamination can cause unexpected bands. Include a control lane without primary antibody to identify any bands arising from the secondary antibody alone.

For accurate interpretation, remember that most commercial antibodies report the observed molecular weight of RNMT as approximately 55-60 kDa .

What factors might affect the subcellular localization of RNMT observed in immunofluorescence?

When studying RNMT localization using immunofluorescence, several factors can influence the observed pattern:

• Fixation method: Different fixation protocols can affect epitope accessibility and preservation of subcellular structures. RNMT is primarily a nuclear protein, but improper fixation may lead to diffuse staining or artifactual cytoplasmic signal.

  • Paraformaldehyde (4%) is commonly used for general fixation

  • Methanol fixation may better preserve nuclear structure but can denature some epitopes

• Cell cycle stage: RNMT distribution may vary during different cell cycle phases, particularly as transcription and mRNA processing dynamics change. Synchronizing cells or co-staining with cell cycle markers can help interpret heterogeneous staining patterns.

• Experimental treatments: Treatments affecting transcription, such as DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), can alter RNMT recruitment to transcription sites .

• Antibody specificity: Ensure the antibody recognizes the intended RNMT epitope. The N-terminal domain (amino acids 1-120) and catalytic domain (amino acids 121-476) may show different localization patterns .

• Detection method: Signal amplification methods and microscopy settings can influence the apparent distribution. Use consistent imaging parameters when comparing conditions.

• Expression system: When studying tagged constructs, overexpression can sometimes lead to aberrant localization. Compare endogenous RNMT with tagged versions to identify potential artifacts.

Studies have shown that while RNMT is predominantly nuclear, its recruitment to specific transcription initiation sites is mediated by its N-terminal domain in a DRB-dependent manner .

How should I interpret changes in RNMT expression levels in response to cellular stimuli?

Interpreting changes in RNMT expression requires careful consideration of several factors:

• Normalization approach: When quantifying RNMT expression changes by Western blot, use appropriate loading controls:

  • Housekeeping proteins (β-actin, GAPDH, tubulin) for whole cell lysates

  • Nuclear markers (Lamin B1, Histone H3) for nuclear fractions

• Time-course analysis: RNMT upregulation has been observed during specific cellular processes, such as T cell activation . Conducting time-course experiments can reveal the temporal dynamics of RNMT expression changes.

• Post-translational regulation: Changes in RNMT activity may occur independently of expression level changes. Consider complementing expression analysis with activity assays.

• Coordinate regulation: RNMT often functions in complex with RAM. Analyzing both proteins can provide insight into whether the complete methyltransferase complex is regulated coordinately.

• Functional consequences: Correlate RNMT expression changes with downstream effects:

  • mRNA cap methylation levels

  • Translation efficiency

  • Expression of targets like terminal polypyrimidine tract (TOP) mRNAs

• Cell-type specificity: RNMT regulation may differ between cell types. In T cells, RNMT induction by TCR stimulation coordinates mRNA, snoRNA, and rRNA production required for ribosome biogenesis .

When interpreting RNMT upregulation during T cell activation, researchers have linked this to increased ribosome biogenesis, demonstrating that RNMT enhances ribosome abundance and coordinates mRNA capping and processing with increased translational capacity .