TRMT112 Antibody

What is TRMT112 and why is it a significant research target?

TRMT112 is a small evolutionary conserved protein that serves as a co-factor and activator for different methyltransferases (MTases) involved in rRNA, tRNA, and protein methylation. Its significance stems from its role as a central "hub" protein that regulates the activity of at least seven different MTases in mammalian cells. The TRMT112 level and localization in cells are tightly regulated through a mutual feedback loop between TRMT112 and its partner proteins . This regulatory role makes it an important target for understanding fundamental cellular processes involving RNA and protein modification, which influences translation and other key cellular functions.

What types of TRMT112 antibodies are available for research applications?

There are several types of TRMT112 antibodies available for research, primarily polyclonal antibodies derived from rabbit hosts. Commercial options include:

• Polyclonal antibodies suitable for Western blot (WB) and immunohistochemistry (IHC) applications with varying recommended dilutions (WB: 1:500-1:3000; IHC: 1:250-1:1000)

• Affinity-isolated antibodies in buffered aqueous glycerol solutions, validated for immunoblotting (0.04-0.4 μg/mL) and immunohistochemistry (1:50-1:200)

• Antibodies validated against specific cell lines such as HEK-293, HeLa, K-562, PC-3, and SH-SY5Y cells

These antibodies typically recognize human TRMT112, with some also showing reactivity with mouse and rat TRMT112 .

What is the subcellular localization of TRMT112 and how does this affect antibody selection?

Endogenous TRMT112 protein is localized in both the cytoplasm and nucleus in human cells (U2OS cells were specifically studied). This dual localization pattern is important to consider when selecting appropriate antibodies and fixation methods . When using TRMT112 antibodies for immunofluorescence studies, it's essential to select antibodies that can effectively penetrate both cellular compartments and maintain specificity in different subcellular environments. Additionally, researchers should be aware that TRMT112's binding partners show varied localization patterns - some partner methyltransferases like EGFP-N6AMT1 and EGFP-METTL5 distribute throughout the cell, while others like EGFP-WBSCR22 and EGFP-THUMPD2 display strong nuclear signals with WBSCR22 specifically accumulating in the nucleolus . For comprehensive studies, antibodies should be validated for both cytoplasmic and nuclear detection.

How can I design experiments to study the interaction between TRMT112 and its partner methyltransferases?

To study interactions between TRMT112 and partner methyltransferases, several complementary approaches can be implemented:

• Co-immunoprecipitation coupled with SILAC: This approach was successfully used to identify TRMT112-interacting proteins. First, generate stable cell lines expressing tagged TRMT112 (consider C-terminal tagging as N-terminal tags may disrupt interaction surfaces). Then perform SILAC-based co-immunoprecipitation followed by mass spectrometry analysis to identify interacting partners .

• Recombinant protein expression systems: Express EGFP-fusion proteins of suspected MTase partners and perform immunoprecipitation analysis using GFP-Trap systems. This allows verification of interactions with endogenous TRMT112 .

• Dual expression system: Co-express TRMT112 with tagged versions of partner MTases. For example, researchers have successfully used TRMT112-EGFP fusion with E2 epitope-tagged MTases to confirm interactions through co-immunoprecipitation .

• Subcellular co-localization analysis: Overexpress EGFP-fused MTases in cells and counterstain with TRMT112 antibodies to determine whether they co-localize in the same cellular compartments, providing supporting evidence for potential interactions .

These methods should be combined with proper controls, including mutation analysis of the interaction surfaces to validate specificity.

What experimental approaches can determine if TRMT112 affects the stability of its methyltransferase partners?

To investigate TRMT112's effect on partner stability, implement these methodological approaches:

• Co-expression studies with quantitative analysis: Express methyltransferases with and without TRMT112 and compare expression levels using Western blot analysis. Previous studies demonstrated that co-expression with TRMT112 enhanced the expression level of all EGFP-MTases to varying degrees .

• Flow cytometry quantification: For GFP-tagged constructs, measure the mean fluorescent intensity (MFI) of cells expressing the methyltransferase alone versus co-expression with TRMT112. Calculate the activation ratio (MFI with TRMT112 / MFI without TRMT112) to quantify the stabilization effect .

• Dose-dependent studies: Transfect cells with increasing amounts of TRMT112 expression constructs and monitor the levels of endogenous and recombinant methyltransferases to identify threshold effects and regulatory feedback mechanisms .

• Pulse-chase experiments: To directly measure protein stability, perform pulse-chase experiments with cycloheximide treatment to block new protein synthesis, then monitor degradation rates of the methyltransferase in the presence or absence of TRMT112.

• Proteasome inhibition studies: Compare the accumulation of methyltransferases with or without TRMT112 in the presence of proteasome inhibitors to determine if TRMT112 prevents proteasomal degradation.

These approaches should include appropriate controls, such as unrelated proteins that do not interact with TRMT112, to establish specificity.

How can I design experiments to identify RNA substrates of TRMT112-associated methyltransferase complexes?

To identify RNA substrates of TRMT112-associated methyltransferase complexes, implement these sophisticated approaches:

• Cross-linking-assisted immunoprecipitation (CLIP) with high-throughput sequencing: Incorporate 4-thiouridine to facilitate cross-linking between the methyltransferase complex and its RNA targets. Immunoprecipitate the complex using antibodies against TRMT112 or its partner methyltransferase, then perform high-throughput sequencing of bound RNAs. This approach has successfully identified 18S rRNA as a substrate for the METTL5-TRMT112 complex .

• Methyltransferase assays with isotope-labeled SAM: Perform in vitro methyltransferase assays using purified TRMT112-methyltransferase complexes and potential RNA substrates with deuterated S-adenosyl methionine (SAM). Analyze methylated nucleosides by LC-MS/MS to detect specific methylation events, such as the N6-methyladenosine (m6A) modification catalyzed by the METTL5-TRMT112 complex .

• Comparative RNA modification analysis: Extract RNA from wild-type and TRMT112-knockout or methyltransferase-knockout cells, then perform RNA modification analysis using techniques like mass spectrometry to identify differences in modification patterns. The observation that RNA from METTL5-KO cells accumulates approximately double the amount of d3m6A compared to wild-type cells after in vitro methylation supports this approach .

• Targeted mutagenesis of catalytic domains: Create methyltransferase mutants with altered catalytic motifs, then perform RNA binding and methylation assays to correlate RNA binding with enzymatic activity, helping distinguish direct substrates from non-specific binding interactions.

These approaches should be complemented with validation experiments, such as site-specific analysis of identified modification sites using techniques like SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography).

What are the optimal conditions for using TRMT112 antibodies in Western blot applications?

For optimal Western blot results with TRMT112 antibodies, consider these methodological details:

• Sample preparation:

  • TRMT112 is a small protein (approximately 14 kDa observed molecular weight) , requiring appropriate gel percentage (12-15% SDS-PAGE) for optimal resolution

  • Include both cytoplasmic and nuclear fractions in your analysis since TRMT112 is present in both compartments

  • Use fresh samples with protease inhibitors to prevent degradation

• Antibody dilutions:

  • Start with the manufacturer's recommended dilution range (1:500-1:3000 for polyclonal antibodies)

  • For immunoblotting with affinity-isolated antibodies, use 0.04-0.4 μg/mL

  • Optimize through titration experiments for your specific cell types

• Detection considerations:

  • Be aware that recombinant tagged TRMT112 can affect endogenous TRMT112 levels - this is a biological effect rather than an antibody artifact

  • When analyzing TRMT112 co-expressed with its methyltransferase partners, note that expression levels of both proteins may be altered due to stabilization effects

  • Include appropriate controls: positive controls (HEK-293, HeLa, K-562, PC-3, or SH-SY5Y cells) and negative controls (TRMT112 knockout cells if available)

• Troubleshooting:

  • For weak signals, extend exposure time or increase antibody concentration

  • For high background, increase blocking time or washing steps

  • For multiple bands, verify specificity using knockout controls or competing peptides

How can I optimize immunohistochemistry protocols for TRMT112 antibodies?

To optimize immunohistochemistry protocols with TRMT112 antibodies, follow these methodological guidelines:

• Antigen retrieval methods:

  • Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for antigen retrieval, as both have been recommended for TRMT112 antibodies

  • Optimize retrieval time and temperature through systematic testing

• Antibody dilution optimization:

  • Begin with manufacturer-recommended dilutions (1:250-1:1000) or (1:50-1:200) depending on the antibody

  • Perform a dilution series to determine optimal signal-to-noise ratio for your specific tissue

  • Consider tissue-specific optimization, as antibody performance may vary between tissue types

• Detection system selection:

  • For low expression levels, consider using amplification systems like tyramide signal amplification

  • For dual/multiple labeling, select compatible detection systems that avoid cross-reactivity

• Controls and validation:

  • Include positive control tissues (human ovary tumor tissue has been validated)

  • Use blocking peptides to confirm specificity

  • Consider parallel staining with different TRMT112 antibodies recognizing different epitopes

• Counterstaining considerations:

  • When studying TRMT112 co-localization with partner methyltransferases, consider double immunofluorescence approaches

  • Select nuclear counterstains that don't interfere with nuclear TRMT112 detection

Each new tissue type may require protocol adjustments to achieve optimal results.

What are the critical factors to consider when studying TRMT112 in protein-protein interaction experiments?

When studying TRMT112 interactions, consider these critical methodological factors:

• Tag position and selection:

  • Place epitope tags at the C-terminus of TRMT112, as N-terminal tagging can disrupt interaction surfaces with methyltransferase partners

  • Be aware that EGFP fusion to TRMT112 may affect protein stability, as TRMT112-EGFP fusion was observed to be less stable than other tagged versions, resulting in degradation products

• Expression level considerations:

  • The amount of TRMT112 in cells appears to be tightly regulated - overexpression of recombinant TRMT112 decreases endogenous TRMT112 levels

  • There exists a mutual regulation mechanism between TRMT112 and its partner methyltransferases, where co-expression enhances stability of both proteins

• Interaction validation approaches:

  • Use multiple complementary methods to confirm interactions (co-IP, yeast two-hybrid, proximity ligation assay)

  • Consider the strength of interaction - some methyltransferases may have stronger affinity for TRMT112 than others

  • Test interaction in multiple cell types as expression levels of competing binding partners may vary

• Buffer and experimental conditions:

  • Optimize lysis conditions to maintain interactions while effectively solubilizing membrane-associated complexes

  • Consider the potential effect of post-translational modifications on interaction strength

  • Test interaction stability under different salt concentrations and detergent conditions

Understanding these factors will help design robust experiments and correctly interpret results when studying TRMT112 protein-protein interactions.

How can I distinguish between direct and indirect interactions in TRMT112 co-immunoprecipitation experiments?

Distinguishing direct from indirect interactions in TRMT112 co-IP experiments requires methodological rigor:

• In vitro binding assays with purified components:

  • Express and purify recombinant TRMT112 and its potential partner proteins

  • Perform direct binding assays using techniques like surface plasmon resonance or microscale thermophoresis

  • Compare binding affinities between different partners to establish relative interaction strengths

• Crosslinking approaches:

  • Use protein crosslinking with short spacer-arm crosslinkers that can only bridge directly interacting proteins

  • Analyze crosslinked products by mass spectrometry to identify direct binding interfaces

  • Compare crosslinking patterns in cellular contexts versus reconstituted systems

• Mutational analysis of interaction surfaces:

  • Create point mutations on the TRMT112 surface that might disrupt specific interactions

  • Previous studies have shown that single amino acid mutations on the surface of TRMT112 revealed differences in how it interacts with various partner methyltransferases

  • If mutation of a specific residue disrupts interaction with one partner but not others, this supports direct and specific binding

• Competition assays:

  • Express increasing amounts of one partner to see if it competes with and reduces binding of another partner

  • This approach can help establish whether different methyltransferases bind to the same or different surfaces of TRMT112

These approaches, especially when used in combination, can help establish a hierarchy of direct versus indirect interactions in the TRMT112 interactome.

What controls are necessary when analyzing the effects of TRMT112 on methyltransferase expression and stability?

When analyzing TRMT112's effects on methyltransferase expression and stability, implement these essential controls:

• Expression vector controls:

  • Include GFP-only controls when using GFP-fusion proteins to establish baseline expression and stability

  • Previous research established that TRMT112 did not affect the expression level of EGFP alone, providing a neutral baseline for comparison

  • Use the same promoter and vector backbone for all constructs to ensure comparable expression

• Protein stability controls:

  • Include both stabilized (known TRMT112 partners) and non-stabilized proteins (non-partners) as positive and negative controls

  • Analyze multiple timepoints after expression to distinguish between effects on transcription, translation, and protein stability

  • Use proteasome inhibitors to determine if stability effects are proteasome-dependent

• Specificity controls:

  • Test structurally similar but functionally distinct proteins to confirm specificity of the stabilization effect

  • Create TRMT112 interface mutants that disrupt specific interactions to confirm dependence on direct binding

  • Use siRNA or CRISPR to deplete endogenous TRMT112 and assess effects on partner stability

• Dosage controls:

  • Perform titration experiments with varying ratios of TRMT112 to methyltransferase

  • Research has shown that increasing the amount of exogenous TRMT112 above certain levels causes endogenous TRMT112 to decrease, indicating complex regulatory mechanisms

• Cell type controls:

  • Test effects in multiple cell lines to ensure the stability effect is not cell-type specific

  • Consider cells with different basal expression levels of TRMT112 and its partners

These controls help distinguish specific biological effects from experimental artifacts and provide mechanistic insights into TRMT112's role as a stability factor.

How should methyltransferase activity data be normalized when studying TRMT112-dependent complexes?

Proper normalization of methyltransferase activity data for TRMT112-dependent complexes requires careful methodological consideration:

• Protein amount normalization:

  • Normalize activity based on the limiting component of the complex

  • When comparing TRMT112-bound versus unbound methyltransferase activity, account for stability differences by using equal amounts of active enzyme rather than equal total protein

  • Consider using quantitative Western blots to determine the actual amount of active enzyme complex

• Activity controls:

  • Include known substrates with established methylation sites as positive controls

  • Use enzyme variants with catalytic mutations as negative controls

  • Consider time-course experiments to ensure measurements are taken in the linear range of activity

• Complex formation considerations:

  • Verify complex formation before activity measurements using techniques like gel filtration or native PAGE

  • For the METTL5-TRMT112 complex, mutation of the METTL5 catalytic motif affects activity but still allows complex formation, providing important mechanistic insights

  • Pre-form complexes under controlled conditions to ensure consistent complex formation

• Substrate normalization:

  • For RNA methylation studies, account for pre-existing methylation in substrates

  • When using cellular RNA, the methylation background may differ between samples - RNA from METTL5-KO cells accumulated approximately double the amount of d3m6A compared to WT cells in in vitro assays

  • Consider using synthetic substrates with defined modification states for precise activity measurements

• Data presentation:

  • Report both absolute activity and fold-change relative to controls

  • Present paired data for methyltransferase with and without TRMT112 to highlight dependency

  • Consider multivariate analysis when comparing across different methyltransferases with varying TRMT112 dependencies

These normalization approaches ensure accurate comparison of enzymatic activities across experimental conditions and between different TRMT112-dependent methyltransferase complexes.

What are the methodological considerations for studying the role of TRMT112 in translational regulation through 18S rRNA methylation?

When investigating TRMT112's role in translational regulation through the METTL5-TRMT112 complex's activity on 18S rRNA, consider these methodological approaches:

• Ribosome profiling experiments:

  • Compare ribosome occupancy and translation efficiency in wild-type versus TRMT112 or METTL5 knockout/knockdown cells

  • Correlate changes in translation with alterations in 18S rRNA methylation status

  • Focus analysis on specific mRNA cohorts that might be differentially affected

• Site-specific methylation analysis:

  • Use primer extension assays, mass spectrometry, or specific antibodies to quantify methylation at the precise target site in 18S rRNA

  • The METTL5-TRMT112 complex specifically catalyzes N6-methyladenosine (m6A) formation in 18S rRNA, which can be detected through specialized mass spectrometry approaches

  • Compare methylation levels across different cellular conditions and in response to various stresses

• Structural studies of the ribosome:

  • Investigate how 18S rRNA methylation affects ribosome structure using cryo-EM

  • Compare structures of ribosomes from wild-type and methylation-deficient cells

  • Focus on regions where the methylation occurs to identify potential effects on ribosome-mRNA or ribosome-tRNA interactions

• Translational fidelity assays:

  • Determine whether loss of 18S rRNA methylation affects translation start site selection, readthrough, or error rates

  • Use reporter constructs with programmed frameshifts or premature termination codons to measure translational fidelity

• Polysome analysis:

  • Compare polysome profiles between wild-type and TRMT112/METTL5-deficient cells

  • Isolate translated mRNAs from different polysome fractions and analyze them by RNA-seq to identify differentially translated transcripts

These methodological approaches can help establish the mechanistic link between TRMT112-dependent 18S rRNA methylation and specific aspects of translational regulation.

How can researchers investigate potential crosstalk between different TRMT112-methyltransferase complexes?

To investigate crosstalk between different TRMT112-methyltransferase complexes, implement these sophisticated methodological approaches:

• Competitive binding studies:

  • Express multiple methyltransferase partners at varying ratios to determine if they compete for limited TRMT112

  • Use tagged versions of different methyltransferases to track their association with TRMT112 in the presence of competitors

  • Perform sequential immunoprecipitations to determine if TRMT112 simultaneously binds multiple partners or forms distinct complexes

• Cellular compartment-specific analysis:

  • Since TRMT112 partners show different subcellular localizations (nuclear vs. cytoplasmic), use compartment-specific isolation techniques to determine the distribution of TRMT112 complexes

  • Compare the composition of TRMT112 complexes in nuclear versus cytoplasmic fractions

  • Use proximity ligation assays to visualize and quantify specific TRMT112-methyltransferase interactions in different cellular locations

• Modification crosstalk analysis:

  • Determine if depletion of one TRMT112 partner affects the activity or substrate modification patterns of other partners

  • Create cellular systems where individual methyltransferase partners can be selectively depleted or inhibited

  • Perform global analysis of RNA and protein methylation patterns to identify compensatory or interdependent modifications

• Developmental and tissue-specific expression analysis:

  • Compare the expression ratios of TRMT112 and its various partners across different tissues and developmental stages

  • Identify conditions where the TRMT112:partner ratio changes dramatically, potentially forcing competition

  • Correlate these expression patterns with functional outcomes in terms of substrate methylation

• Dynamics of complex formation:

  • Use real-time imaging techniques like FRAP (Fluorescence Recovery After Photobleaching) to measure the dynamics of TRMT112 association with different partners

  • Employ methods like FLIM-FRET to measure interaction strength between TRMT112 and its partners in living cells

  • Investigate whether cellular stress or signaling events cause redistribution of TRMT112 between different partner complexes

These approaches will help establish whether different TRMT112-methyltransferase complexes function independently or exhibit regulatory crosstalk through competition for limited TRMT112.

What technical approaches can help elucidate the structural basis of TRMT112 interaction with multiple methyltransferase partners?

To elucidate the structural basis of TRMT112's interactions with multiple methyltransferase partners, consider these advanced technical approaches:

• Comparative structural analysis:

  • Perform X-ray crystallography or cryo-EM studies of TRMT112 in complex with different partner methyltransferases

  • Compare binding interfaces to identify common and distinct interaction motifs

  • Research has shown that while TRMT112 interacts with its partners in a similar way, single amino acid mutations on the surface of TRMT112 reveal several differences in interaction patterns

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Use HDX-MS to map the binding interfaces between TRMT112 and different partners

  • Compare protection patterns to identify common and partner-specific protected regions

  • This technique can provide insights into dynamics of interaction that may not be captured by static structural methods

• Systematic mutagenesis with functional readouts:

  • Create a comprehensive library of TRMT112 surface mutations

  • Test each mutant for interaction with all known partner methyltransferases

  • Develop interaction fingerprints for each partner based on mutational sensitivity patterns

• Computational modeling and molecular dynamics:

  • Use available structural data to model TRMT112 interactions with partners where co-crystal structures aren't available

  • Perform molecular dynamics simulations to understand the flexibility and adaptability of interaction interfaces

  • Identify potential allosteric effects that might influence binding to different partners

• Cross-linking mass spectrometry:

  • Use chemical cross-linking followed by mass spectrometry to identify residues in close proximity at protein-protein interfaces

  • Compare cross-linking patterns between different TRMT112-methyltransferase complexes

  • This approach can provide distance constraints for structural modeling

• Single-molecule techniques:

  • Apply single-molecule FRET or force spectroscopy to measure interaction dynamics and strength

  • Compare binding kinetics and thermodynamics for different TRMT112-methyltransferase pairs

  • These approaches can reveal potential conformational changes upon binding

These technical approaches, especially when used in combination, can provide comprehensive insights into how TRMT112 achieves specific recognition of multiple methyltransferase partners while maintaining selectivity.