TRMT6 Antibody

What is TRMT6 and what are its fundamental functions?

TRMT6 (tRNA methyltransferase 6) is a 497 amino acid protein that functions as the substrate-binding subunit of tRNA (adenine-N(1)-)-methyltransferase. It forms a heterodimer with TRMT61A (also known as TRM61) and catalyzes the formation of N(1)-methyladenine at position 58 (m1A58) in initiator methionyl-tRNA . TRMT6 localizes primarily to the nucleus and is expressed in various tissues including liver, brain, ovary, and testis .

The protein plays a critical role in tRNA modifications that regulate the efficiency of mRNA translation by maintaining correct reading frames. TRMT6 has a calculated molecular weight of 56 kDa, though it is typically observed at 55-60 kDa in experimental conditions . The protein's full name is tRNA methyltransferase 6 homolog (S. cerevisiae), and it is also known by several aliases including GCD10, TRM6, and MGC5029 .

What applications can TRMT6 antibodies be validated for?

TRMT6 antibodies can be validated for multiple research applications as demonstrated by commercial antibodies:

It is strongly recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the optimal dilution can be sample-dependent . For IHC applications, antigen retrieval with TE buffer pH 9.0 is typically suggested, though citrate buffer pH 6.0 may be used alternatively .

How should researchers approach validation of TRMT6 antibodies?

Proper validation of TRMT6 antibodies requires a multi-faceted approach to ensure specificity and reliability. Researchers should begin with Western blot analysis to confirm that the antibody detects a protein of the expected molecular weight (55-60 kDa) . This validation should include positive controls (tissues or cell lines known to express TRMT6, such as U-251, HepG2, or mouse brain tissue) and negative controls (samples with TRMT6 knockdown) .

For advanced validation, siRNA-mediated knockdown of TRMT6 is particularly valuable. The experimental design should include:

• Transfection with specific siRNAs targeting TRMT6 (e.g., Horizon Dharmacon, #L-017324-02-0005) at 10nM final concentration

• Use of appropriate transfection reagents such as LipofectamineTM RNAiMAX

• Collection of cells 96 hours after first transfection (with double transfection protocol)

• Parallel transfection with negative control siRNAs

• Analysis of knockdown efficiency by both qRT-PCR and Western blot

Additional validation through orthogonal methods such as IHC or IF/ICC in tissues with known TRMT6 expression patterns further strengthens confidence in antibody specificity .

What are the optimal storage and handling conditions for TRMT6 antibodies?

For maximum stability and performance, TRMT6 antibodies should be stored according to manufacturer specifications. Commercial TRMT6 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . The recommended storage temperature is -20°C, where the antibody generally remains stable for one year after shipment .

Important handling considerations include:

• Avoiding repeated freeze-thaw cycles by making single-use aliquots upon receipt

• Maintaining sterile conditions during handling to prevent contamination

• Keeping the antibody on ice during experimental procedures

• For small volume antibodies (20μl), note that some formulations may contain 0.1% BSA

• Ensuring proper temperature control during shipping and receiving

Aliquoting is generally unnecessary for -20°C storage according to manufacturer guidelines, but may be beneficial for antibodies frequently accessed for experiments .

How does TRMT6-mediated m1A modification impact cancer progression?

TRMT6-mediated N1-methyladenosine (m1A) modification has been identified as a significant epigenetic mechanism influencing tumor progression across multiple cancer types. In colorectal cancer (CRC), higher m1A methylation levels have been detected compared to normal tissues . Research has demonstrated that high TRMT6 expression significantly correlates with advanced tumor stages (III&IV vs. I&II, p<0.05) and poor prognosis, with 5-year relapse-free survival rates of 50.9% versus 65.1% in high versus low expression groups (p<0.05) .

Mechanistically, TRMT6 appears to exert its oncogenic effects through several pathways:

• Enhancement of cancer stem cell (CSC) properties: TRMT6 has been found to be required for self-renewal of cancer stem cells

• Promotion of EGFR/ERK signaling: Studies have elucidated that TRMT6-mediated m1A modification operates through the EGFR/ERK pathway

• Cellular proliferation: Depletion of TRMT6/TRMT61A reduces proliferation capacity in bladder cancer cell lines (5637 and HT1197)

• Cell migration and displacement: Knockdown of TRMT6/TRMT61A decreases average cell displacement in certain cancer cell lines

These findings collectively suggest that TRMT6-mediated m1A modifications represent a potential therapeutic target and prognostic marker in various cancers .

What is the relationship between TRMT6 expression and cellular stress responses?

TRMT6 appears to play a significant role in cellular stress response mechanisms, particularly in relation to the unfolded protein response (UPR). Research on bladder cancer (BLCA) cell lines has revealed interesting connections between TRMT6/TRMT61A function and stress tolerance:

• TRMT6/TRMT61A depletion decreases mRNA expression of targets associated with the ATF6-branch of the UPR in certain cell lines (e.g., 5637) but not others (e.g., HT1197)

• Cell survival after induction of cellular stress (such as tunicamycin treatment) is compromised following TRMT6/TRMT61A knockdown in some cancer cell lines

• The relationship between tunicamycin concentration, cell line, and cell viability shows statistical significance in ANOVA analysis, suggesting TRMT6 plays a role in modulating cellular responses to stress

These findings indicate that TRMT6 is involved in desensitizing cancer cells against cellular stress, potentially contributing to cancer cell survival under adverse conditions. This relationship between TRMT6 and stress response pathways may provide novel insights for cancer treatment strategies .

What methodological approaches are recommended for investigating TRMT6/TRMT61A complex function?

Investigating the TRMT6/TRMT61A complex requires specialized methodological approaches that address both the individual proteins and their interactions. Based on current research methodologies, the following approaches are recommended:

• Co-immunoprecipitation (Co-IP): To confirm the interaction between TRMT6 and TRMT61A

  • Use anti-TRMT6 antibodies (e.g., Abcam #ab235321) for immunoprecipitation

  • Detect TRMT61A (e.g., using Biorbyt #orb411814) in the precipitated complex

  • Include appropriate controls (IgG control, input samples)

• Dual knockdown experiments:

  • Transfect cells with siRNAs targeting TRMT6 (e.g., Horizon Dharmacon, #L-017324-02-0005) and TRMT61A (e.g., Horizon Dharmacon, #L-015870-01-0005)

  • Implement double transfection protocol with 10nM final siRNA concentration

  • Collect cells 96 hours post first transfection for optimal knockdown

• m1A methylation analysis:

  • Utilize m1A dot-blot assays to detect methylation levels in tissues and cell lines

  • Compare methylation levels between normal and cancer tissues

  • Correlate methylation levels with TRMT6/TRMT61A expression

• Functional readouts:

  • Cell viability assays (e.g., Prestoblue Cell Viability Reagent)

  • Wound-healing assays for migration assessment

  • Live-cell imaging-based cell displacement analysis

The combination of these approaches provides a comprehensive assessment of TRMT6/TRMT61A function in both normal and pathological contexts.

How should researchers troubleshoot non-specific binding when using TRMT6 antibodies?

Non-specific binding is a common challenge when working with antibodies, including those targeting TRMT6. A systematic troubleshooting approach includes:

• Optimization of antibody dilution:

  • Begin with the manufacturer's recommended range (e.g., 1:2000-1:16000 for WB)

  • Perform a dilution series to identify optimal signal-to-noise ratio

• Blocking optimization:

  • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

  • Extend blocking time to 1-2 hours at room temperature

• Washing protocol refinement:

  • Increase washing steps (5-6 washes of 5-10 minutes each)

  • Add 0.05-0.1% Tween-20 to washing buffer (PBST)

• Positive and negative controls:

  • Include cell lines with known TRMT6 expression (U-251, HepG2, U-87 MG, SMMC-7721, HuH-7)

  • Incorporate TRMT6 knockdown samples as negative controls

• Detection system adjustment:

  • For Western blot, use appropriate detection reagents based on target abundance (SuperSignal West Femto Maximum Sensitivity Substrate for TRMT6)

  • Ensure secondary antibody species compatibility and dilution optimization

If non-specific binding persists, cross-validation with an alternative TRMT6 antibody from a different manufacturer or different clone/lot number may help confirm results.

What are the optimal protocols for Western blot analysis of TRMT6?

Optimal Western blot analysis of TRMT6 requires careful attention to sample preparation, antibody selection, and detection protocols. Based on successful experimental approaches, the following protocol is recommended:

Sample preparation:

• Extract total protein using standard lysis buffers (RIPA or NP-40 with protease inhibitors)

• Determine protein concentration using Bradford or BCA assay

• Load 20-30 μg protein per lane

• Denature samples at 95°C for 5 minutes in Laemmli buffer with reducing agent

Gel electrophoresis and transfer:

• Separate proteins on 10-12% SDS-PAGE gel (TRMT6 observed MW: 55-60 kDa)

• Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes

Antibody incubation:

• Block membrane for 1 hour in 5% milk in PBS with 0.05% Tween-20 (PBST)

• Incubate with anti-TRMT6 primary antibody (e.g., Abcam #ab235321, 1:1000 dilution) overnight at 4°C

• Wash 3-5 times with PBST

• Incubate with appropriate HRP-linked secondary antibody (e.g., anti-rabbit, GE Healthcare Life Sciences #NA934-100UL, 1:10,000) for 1 hour at room temperature

Detection and analysis:

• Develop using enhanced chemiluminescence (e.g., SuperSignal West Femto Maximum Sensitivity Substrate)

• Image using a digital imaging system (e.g., Biorad ChemiDoc XRS+)

• Quantify band intensities with appropriate software (e.g., ImageLab)

• Normalize to loading controls such as GAPDH (Abcam, #ab125247, 1:3000)

This protocol has been validated in multiple cell lines including U-251, HepG2, U-87 MG, SMMC-7721, and HuH-7 .

How should researchers design experiments to investigate TRMT6's role in cancer progression?

Designing experiments to investigate TRMT6's role in cancer progression requires a multi-faceted approach that incorporates molecular, cellular, and clinical analyses:

• Expression analysis in patient cohorts:

  • Compare TRMT6 expression in paired tumor and adjacent-normal tissues

  • Correlate expression with clinical parameters (tumor stage, patient survival)

  • Analyze public databases (TCGA, GEO) and validate findings in independent cohorts

• Functional studies through gene manipulation:

  • Implement TRMT6 knockdown using siRNA or shRNA approaches

  • Assess effects on:

    • Cell viability and proliferation

    • Colony formation

    • Spheroid formation (for cancer stem cell properties)

    • Migration (wound healing assays)

    • Invasion assays

• Mechanistic investigations:

  • Analyze m1A methylation levels using dot-blot assays

  • Perform RNA-sequencing after TRMT6 depletion to identify affected pathways

  • Investigate specific signaling pathways (e.g., EGFR/ERK) through Western blot analysis of phosphorylated proteins

• Stress response studies:

  • Challenge cells with stress inducers (e.g., tunicamycin)

  • Compare survival between control and TRMT6-depleted cells

  • Analyze UPR pathway components (ATF6-branch)

• In vivo validation:

  • Develop xenograft models with TRMT6-depleted cancer cells

  • Monitor tumor growth, metastasis, and response to therapy

  • Correlate findings with in vitro observations

This comprehensive approach enables researchers to establish both correlative and causative relationships between TRMT6 function and cancer progression.

What considerations are important when studying TRMT6 across different cancer types?

When investigating TRMT6 across different cancer types, several important considerations must be addressed to ensure comprehensive and accurate analyses:

• Baseline expression variability:

  • TRMT6 expression varies significantly across cancer types and even within cancer subtypes

  • Heterogeneous morphology, proliferation, displacement, and stress sensitivity have been observed among different cancer cell line panels

  • Appropriate normal tissue controls specific to each cancer type are essential

• Cell line selection:

  • Include multiple cell lines representing the diversity within each cancer type

  • For bladder cancer studies, cell lines such as 5637 and HT1197 have been successfully used

  • For colorectal cancer, RKO and SW620 cell lines have been employed

  • Include SV-HUC-1 as a normal urinary tract epithelium control for bladder cancer studies

• Cancer-specific pathway interactions:

  • The signaling pathways affected by TRMT6 may differ between cancer types

  • In colorectal cancer, EGFR/ERK signaling has been implicated

  • In bladder cancer, connections to the ATF6-branch of the UPR have been observed

• Differential responses to TRMT6 depletion:

  • Not all cancer cell lines respond identically to TRMT6 knockdown

  • Cell survival after stress induction was compromised in 5637 but not in HT1197 cells following TRMT6/TRMT61A knockdown

  • Design experiments to include multiple outcome measures that capture this heterogeneity

• Clinical correlation approaches:

  • Different cancer types may require distinct clinical correlation metrics

  • For colorectal cancer, relapse-free survival has shown significant correlation with TRMT6 expression

  • Tumor staging correlation may vary by cancer type

These considerations highlight the importance of tailored experimental designs that account for the biological complexity and heterogeneity across different cancer types.

How might TRMT6 serve as a therapeutic target in cancer treatment?

TRMT6's emerging role in cancer progression suggests several promising avenues for therapeutic targeting:

• Direct inhibition of TRMT6/TRMT61A enzymatic activity:

  • Development of small molecule inhibitors targeting the methyltransferase activity

  • Structure-based drug design focusing on the TRMT6/TRMT61A heterodimer interface

  • Screening of compound libraries for molecules that disrupt m1A formation

• Targeting TRMT6-dependent vulnerabilities:

  • Exploiting synthetic lethality approaches by identifying genes/pathways that become essential in TRMT6-overexpressing cancers

  • Combining TRMT6 inhibition with cellular stress inducers, as TRMT6 appears to protect cancer cells from stress-induced death

  • Targeting the ATF6-branch of the UPR in conjunction with TRMT6 inhibition

• Disruption of cancer stem cell maintenance:

  • As TRMT6 enhances stem cell-like properties required for cancer stem cell self-renewal , therapies targeting this specific function could reduce tumor recurrence and metastasis

  • Development of strategies to selectively target TRMT6-dependent stem cell populations

• Modulation of EGFR/ERK signaling:

  • Combination therapies targeting both TRMT6 and components of the EGFR/ERK pathway may provide synergistic effects

  • Exploring TRMT6 as a biomarker for responsiveness to existing EGFR inhibitors

• RNA modification-based therapeutics:

  • Development of therapeutic approaches that selectively modify RNA methylation patterns

  • Application of epitranscriptomic editing tools to reverse pathological m1A patterns

Future investigations should focus on validating these approaches across diverse cancer types and evaluating their efficacy in combination with established therapies.

What emerging technologies might enhance TRMT6 research?

Several cutting-edge technologies hold promise for advancing TRMT6 research:

• Single-cell epitranscriptomics:

  • Application of single-cell sequencing technologies to map m1A modifications at the individual cell level

  • Investigation of heterogeneity in TRMT6 expression and activity within tumors

  • Correlation of single-cell TRMT6 expression with cancer stem cell markers

• CRISPR-based functional genomics:

  • CRISPR-Cas9 screens to identify synthetic lethal interactions with TRMT6

  • CRISPR activation/inhibition systems for precise modulation of TRMT6 expression

  • Base editing approaches for introducing specific mutations in TRMT6

• Advanced RNA modification mapping:

  • Development of high-resolution techniques for mapping m1A modifications in small RNAs

  • Application of nanopore direct RNA sequencing for real-time detection of modified nucleotides

  • Integration of computational approaches for predicting m1A sites and their functional consequences

• Spatial transcriptomics:

  • Investigation of spatial distribution of TRMT6 expression within tumor tissues

  • Correlation of TRMT6 expression with tumor microenvironment features

  • Integration with single-cell data to create comprehensive maps of m1A modification landscapes

• Protein structure determination:

  • Cryo-EM or X-ray crystallography studies of the TRMT6/TRMT61A complex

  • Structural insights to guide rational design of specific inhibitors

  • Investigation of structural changes upon substrate binding or protein-protein interactions

These technological advances will provide unprecedented insights into TRMT6 biology and accelerate the development of therapeutic strategies targeting this important epitranscriptomic regulator.