TRMT5 Antibody

What is TRMT5 and what cellular functions does it perform?

TRMT5 (tRNA methyltransferase 5) is a 509-amino acid protein that methylates the N1 position of guanosine-37 in various tRNAs using S-adenosyl methionine as a methyl donor . This represents the first step in wybutosine biosynthesis, a modified base adjacent to the anticodon of tRNAs required for accurate decoding during translation .

The protein is initially produced in the cytosol and subsequently transported into mitochondria, where it catalyzes the formation of 1-methylguanosine (m1G) at position 37 of mitochondrial tRNAs . This methylation is critical for maintaining the reading frame during peptide elongation, as it protects against +1 frameshifting errors that can cause premature termination of protein synthesis .

What are the optimal applications for TRMT5 antibodies?

TRMT5 antibodies are validated for multiple research applications:

For optimal results, each antibody should be titrated in your specific experimental system, as sample type and detection method can significantly influence performance .

What species reactivity do common TRMT5 antibodies demonstrate?

TRMT5 antibodies show cross-reactivity with multiple species due to sequence conservation:

When working with non-human samples, verification of antibody performance is recommended, as reactivity may vary based on epitope conservation .

What is the observed molecular weight of TRMT5 in experimental conditions?

While the calculated molecular weight of TRMT5 is 58 kDa (509 amino acids), experimental observations show slight variations:

• 18255-1-AP detects TRMT5 at 50-55 kDa

• Other antibodies generally report the expected 58 kDa band

These variations may reflect post-translational modifications, cleavage events, relative charges, or other experimental factors that affect protein migration during electrophoresis .

How can researchers validate TRMT5 antibody specificity in experimental systems?

A comprehensive validation strategy should include:

• Positive and negative cell/tissue controls: Use cells known to express TRMT5 (e.g., Jurkat, MCF-7, A431, NIH/3T3) alongside low-expression or knockout samples

• Knockdown/knockout validation: Compare antibody signal in wild-type versus TRMT5 RNAi or CRISPR-edited cells. Publications demonstrating KD/KO validation are available for some antibodies

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Multiple antibody comparison: Use antibodies targeting different epitopes of TRMT5 to confirm consistent detection

• Recombinant expression validation: Transiently express tagged TRMT5 and confirm co-localization or size-appropriate detection

In subcellular localization studies, mitochondrial fractionation experiments with proteinase K treatment can be used to confirm TRMT5 localization, as demonstrated in HeLa cells where endogenous TRMT5 was enriched within the mitochondrial fraction alongside matrix protein mtSSB1 .

What are the pathological implications of TRMT5 mutations and how can antibodies facilitate research in this area?

TRMT5 mutations cause Combined Oxidative Phosphorylation Deficiency 26 (COXPD26), an autosomal recessive disorder characterized by:

• Early-onset symptoms

• Developmental delay

• Gastrointestinal dysfunction

• Exercise intolerance

• Hypotonia and muscle weakness

• Neuropathy

• Spastic diplegia

Specific mutations identified include:

• c.312_315del; c.872G>A

• c.312_315del; c.1156A>G

• c.881A>C (p.E294A)

• c.1218G>C (p.Q406H)

• c.1481C>T (p.T494M)

TRMT5 antibodies can be used to:

• Assess protein levels in patient samples

• Study the impact of mutations on protein localization

• Investigate downstream effects on mitochondrial tRNA methylation

• Evaluate respiratory chain complex assembly and function

When analyzing patient samples, researchers should consider the genotype-phenotype correlation, as clinical presentation varies significantly between patients with different TRMT5 mutations .

What methodologies are optimal for studying TRMT5-mediated tRNA modifications?

A comprehensive approach to studying TRMT5-mediated tRNA modifications includes:

• In vitro methylation assays: Using recombinant TRMT5 and synthetic tRNA substrates to measure m1G37 formation, though these assays have shown lower activity with mitochondrial versus cytosolic substrates

• Mass spectrometry analysis: To quantify m1G37 levels in tRNAs isolated from cells with wild-type or mutant TRMT5

• High-resolution northern blotting: To detect changes in tRNA stability resulting from hypomodification

• tRNA-Seq: For comprehensive analysis of all tRNA modifications in cells with TRMT5 perturbations

• Immunoprecipitation with TRMT5 antibodies: To identify TRMT5-interacting partners involved in tRNA modification pathways

• Heterologous yeast models: Complementation studies using human TRMT5 in yeast with TRM5 deletions can confirm functional conservation and assess pathogenic variants

When designing experiments, consider that approximately 11 types of tRNAs in eukaryotes can be modified with m1G37, with confirmed modifications in mitochondrial tRNALeu(CUN) and tRNAPro .

How does TRMT5 expression correlate with cancer pathology, and what research approaches are recommended?

TRMT5 expression has been investigated across multiple cancer types, as documented in the Human Protein Atlas . Recent research has demonstrated that:

• TRMT5 has been identified as a potential target in hepatocellular carcinoma (HCC), with studies showing that targeting TRMT5 suppresses HCC progression via inhibition of the HIF-1α pathway

• TRMT5 has been identified in integrated feature selection methods as part of gene signatures for ischemic cardiomyopathy

For cancer-related TRMT5 research, recommended approaches include:

• Immunohistochemical analysis: To assess TRMT5 expression levels across tumor types and correlate with clinical parameters

• Functional studies: Using TRMT5 antibodies in combination with knockdown/overexpression systems to investigate its role in cancer cell proliferation, migration, and invasion

• Mechanistic investigations: To elucidate how TRMT5-mediated tRNA modifications might contribute to translational dysregulation in cancer cells

• Correlation with hypoxia markers: Given the connection to HIF-1α pathways, investigating TRMT5 expression in hypoxic tumor regions

When designing cancer-related studies, researchers should consider both TRMT5's enzymatic function and potential non-canonical roles in cellular signaling pathways.

What experimental controls and considerations are critical when studying mitochondrial dysfunction related to TRMT5?

When investigating TRMT5's role in mitochondrial function, several critical controls and considerations should be implemented:

• Mitochondrial isolation quality: Verify purity using markers for different mitochondrial compartments (outer membrane: TOM22; matrix: mtSSB1) and confirm TRMT5 resistance to proteinase K treatment, as demonstrated in previous studies

• Multiple respiratory chain complex assessment: TRMT5 mutations impact multiple respiratory chain complexes, so comprehensive analysis should include:

  • Complex I (NADH:ubiquinone oxidoreductase)

  • Complex III (ubiquinol:cytochrome c oxidoreductase)

  • Complex IV (cytochrome c oxidase)

  • Complex V (ATP synthase)

• Tissue-specific effects: TRMT5-related hypomodification of G37 is particularly prominent in skeletal muscle, suggesting tissue-specific studies are essential

• Biochemical parameters: Monitor relevant biochemical markers observed in COXPD26 patients:

  Parameter	Observed Values
  Glutamic pyruvic transaminase	100.2 (5-35) U/L
  Myoglobin	77.4 (11.6-73.0) ng/ml
  Blood glucose	normal (4.7-7.5 mmol/L)
  Cardiac troponin I	0.08 (0.00-0.09) pg/ml
  Creatine kinase isoenzyme	2.5 (0.0-3.7) ng/ml

• Rescue experiments: Re-expression of wild-type TRMT5 should rescue the molecular phenotype in cells with TRMT5 mutations or knockdown

When interpreting results, consider that clinical presentations of TRMT5 mutations vary significantly despite similar biochemical abnormalities, suggesting complex genotype-phenotype relationships.

What are the optimal storage and handling conditions for TRMT5 antibodies?

For maximum stability and performance of TRMT5 antibodies, follow these storage recommendations:

Critical handling considerations:

• Aliquoting is recommended for antibodies without glycerol to avoid repeated freeze-thaw cycles

• Some formulations contain sodium azide, which is a hazardous substance requiring trained handling

• For antibodies containing 50% glycerol, aliquoting may be optional for storage at -20°C

• Note that glycerol may interfere with some downstream applications and should be added with caution

How should researchers optimize Western blot protocols specifically for TRMT5 detection?

For optimal Western blot detection of TRMT5:

• Sample preparation:

  • Use validated positive controls such as Jurkat cells, MCF-7 cells, mouse testis tissue, or NIH/3T3 cells

  • Load 20-30 μg of total protein per lane (based on successful detection reported for Jurkat whole cell lysate at 20 μg)

• Blocking conditions:

  • 5% non-fat dry milk in TBST has been validated for ab259986

  • Optimize blocking reagent based on specific antibody recommendations

• Antibody dilutions:

  • Primary antibody: Follow recommended dilutions (1:500-1:3000, depending on the antibody)

  • For ab259986, 1:1000 dilution has been validated

  • For NBP1-52876, 1.0 μg/ml is recommended

• Detection sensitivity:

  • Consider enhanced chemiluminescence (ECL) substrate sensitivity; higher sensitivity ECL may be required as demonstrated with ab259986

  • Exposure times may need optimization: 3 minutes standard exposure, but up to 103 seconds with higher sensitivity ECL has been reported

• Expected results:

  • Look for bands between 50-58 kDa

  • Be aware that the observed molecular weight may differ from the calculated 58 kDa due to post-translational modifications

What methodological approaches should be used to investigate TRMT5 interactions with target tRNAs?

To investigate TRMT5-tRNA interactions, researchers should consider these methodological approaches:

• RNA immunoprecipitation (RIP):

  • Use TRMT5 antibodies to pull down TRMT5-tRNA complexes

  • Analyze bound tRNAs through RT-PCR or sequencing

  • Include controls for non-specific RNA binding

• Crosslinking and immunoprecipitation (CLIP):

  • UV crosslinking to capture direct RNA-protein interactions

  • Immunoprecipitate with TRMT5 antibodies

  • Sequence bound tRNAs to identify specific substrates

• In vitro binding assays:

  • Express and purify recombinant TRMT5

  • Assess binding to synthetic tRNA substrates

  • Remember that in vitro methylation assays with human TRMT5 have shown lower activity with mitochondrial versus cytosolic substrates

• Structural studies:

  • Use immunoprecipitated TRMT5 for structural analysis of TRMT5-tRNA complexes

  • Consider that TRMT5 is not highly specific with respect to the structure of bound tRNAs to be methylated

• Methylation site analysis:

  • Focus on guanosine-37 position, which is next to the 3' end of the anticodon

  • Consider that approximately 11 types of tRNAs in eukaryotes can be modified with m1G37

  • Confirmed modifications exist in mitochondrial tRNALeu(CUN) and tRNAPro

How can TRMT5 antibodies contribute to understanding broader mitochondrial disease mechanisms?

TRMT5 research has broader implications for understanding mitochondrial disease mechanisms:

• Linkage to respiratory chain complex assembly:

  • TRMT5 mutations cause multiple respiratory chain complex deficiencies

  • Antibodies can help track how tRNA modifications influence mitochondrial translation efficiency of respiratory complex components

• Tissue-specific effects:

  • TRMT5-related tRNA hypomodification is more prominent in skeletal muscle

  • Antibodies can help elucidate tissue-specific expression patterns and functional consequences

• Integration with other mitochondrial pathways:

  • Use antibodies in co-immunoprecipitation studies to identify TRMT5 interaction partners

  • Investigate relationships between tRNA modifications and other mitochondrial quality control mechanisms

• Therapeutic development:

  • Antibodies can help evaluate the efficacy of experimental therapies aimed at rescuing TRMT5 function

  • Monitor subcellular localization changes in response to treatments

• Biomarker potential:

  • Investigate whether TRMT5 protein levels or localization could serve as biomarkers for mitochondrial dysfunction

  • Correlate with other established markers of mitochondrial disease

What recent advances in cancer research involve TRMT5, and how should research methodologies adapt?

Recent TRMT5 cancer research has yielded significant findings:

• Hepatocellular carcinoma (HCC) progression:

  • Targeting TRMT5 suppresses HCC progression via inhibiting HIF-1α pathways

  • Methodology suggestion: Combine TRMT5 antibodies with HIF-1α pathway markers in multiplexed immunofluorescence studies

• Ischemic cardiomyopathy:

  • TRMT5 has been identified in integrated feature selection methods identifying aging-based gene signatures for ischemic cardiomyopathy

  • Methodology suggestion: Age-stratified analysis of TRMT5 expression in cardiac tissues

• Cancer tissue expression profiling:

  • The Human Protein Atlas has documented TRMT5 expression across 20 different cancer types

  • Methodology suggestion: Correlate TRMT5 protein levels with patient outcomes and treatment responses

Research methodologies should adapt to include:

• Single-cell approaches to detect heterogeneity in TRMT5 expression within tumors

• Integration with "-omics" data to correlate TRMT5 expression with mutation signatures

• Functional studies using CRISPR-based methodologies to precisely modulate TRMT5 in cancer models

• Investigation of potential non-canonical functions beyond tRNA modification

How should researchers approach contradictory data regarding TRMT5 function or localization?

When encountering contradictory data about TRMT5, researchers should systematically:

• Evaluate antibody specificity:

  • Different antibodies target different epitopes, potentially yielding discrepant results

  • Confirm findings using multiple antibodies targeting distinct TRMT5 regions

  • Validate with genetic approaches (siRNA, CRISPR) to confirm specificity

• Consider dual localization:

  • TRMT5 is produced in the cytosol and then transported to mitochondria

  • Cellular fractionation experiments combined with proteinase K treatment can distinguish mitochondrial from cytosolic localization

  • Different fixation methods may affect epitope availability in different cellular compartments

• Assess experimental conditions:

  • Cell type differences: TRMT5 function has been studied in various cell types (HeLa, Jurkat, MCF-7, NIH/3T3)

  • Stress conditions may alter TRMT5 localization or function

  • Growth conditions and cell cycle stage could influence results

• Reconcile in vitro versus in vivo findings:

  • In vitro methylation assays with human TRMT5 showed lower activity with mitochondrial versus cytosolic substrates

  • In vivo, TRMT5 clearly functions in mitochondrial tRNA modification

  • The discrepancy might reflect the need for additional factors in the cellular context

• Consider species differences:

  • While TRMT5 function is broadly conserved, species-specific differences may exist

  • Use heterologous systems (like yeast models) carefully, understanding their limitations