Recombinant Bovine Methyltransferase-like protein 21C (METTL21C)

What is METTL21C and which protein family does it belong to?

METTL21C belongs to the methyltransferase-like 21 (METTL21) family of protein lysine methyltransferases (PKMTs). It is part of a larger superfamily containing more than 100 known and candidate PKMTs in humans. Specifically, METTL21C contains a 7βS domain and is one of four members of the METTL21 subfamily, which also includes METTL21A, METTL21B, and METTL21D. All members of this family catalyze non-histone protein methylation, with each having distinct substrate preferences . Unlike many other methyltransferases, the METTL21 family specifically methylates non-histone proteins and does not target histones, making them important regulators of post-translational modifications in various cellular contexts .

What is the primary tissue distribution of METTL21C expression?

METTL21C exhibits highly tissue-specific expression, being predominantly expressed in skeletal muscle tissue . Studies in chickens have shown that METTL21C is particularly enriched in specific muscle types, including the soleus (SOL) and gastrocnemius (GA) muscles . This muscle-specific expression pattern is consistent across species, suggesting conservation of function. The high expression in skeletal muscle correlates with its functional importance in muscle biology, especially in processes related to myoblast differentiation and muscle development .

What are the confirmed substrates of METTL21C?

Alanine tRNA synthetase 1 (AARS1) has been conclusively identified as a direct substrate of METTL21C through unbiased biochemistry-based screening coupled to mass spectrometry. METTL21C specifically catalyzes mono-, di-, and trimethylation of AARS1 at lysine 943 (AARS1-K943me) both in vitro and in vivo . Additionally, research in chicken myoblasts has identified heat shock cognate 71 kDa protein (Hsc70) as an interacting partner of METTL21C, with evidence suggesting that Hsc70 Lys-561 is a target for METTL21C-mediated methylation . Earlier reports had suggested that valosin-containing protein (VCP) and heat shock 70-kDa protein 8 (HSPA8) might be METTL21C substrates, but more rigorous biochemical analyses found no evidence supporting these claims .

What are recommended methods for detecting METTL21C enzymatic activity in vitro?

To detect METTL21C enzymatic activity in vitro, researchers should consider the following methodological approach:

• Recombinant protein expression: Express and purify recombinant METTL21C protein using bacterial or mammalian expression systems.

• In vitro methylation assay: Incubate purified METTL21C with potential substrates (e.g., AARS1) in the presence of S-adenosyl-L-methionine (SAM) as a methyl donor.

• Detection methods:

  • Radioactive assay: Use [³H]-SAM or [¹⁴C]-SAM and detect methyl transfer by scintillation counting

  • Mass spectrometry: Analyze methylation sites and degrees (mono-, di-, tri-) through LC-MS/MS

  • Antibody-based detection: Use methyl-lysine specific antibodies for Western blot analysis

For rigorous validation, comparison with known METTL21 family members (METTL21A, B, and D) and their respective substrates should be included as controls . When testing novel substrates, include AARS1 as a positive control to confirm enzyme activity .

How can I design a robust siRNA knockdown experiment for METTL21C?

A robust siRNA knockdown experiment for METTL21C should include:

• siRNA design and controls:

  • Design 3-4 different siRNA sequences targeting different regions of METTL21C

  • Include negative control siRNA (non-targeting sequence)

  • Include vehicle control (transfection reagent only)

• Validation of knockdown efficiency:

  • Measure METTL21C mRNA levels via qPCR (72 hours post-transfection is recommended)

  • Confirm protein reduction via Western blot if antibodies are available

  • Aim for at least 70% reduction in expression

• Experimental timeline:

  • For myoblast differentiation studies, transfect cells before inducing differentiation

  • Monitor phenotypic changes at key timepoints (days 3, 5, and 7 for myoblast differentiation)

• Readouts for functional effects:

  • For muscle cells: assess proliferation, differentiation, fusion index, myotube formation

  • For bone cells: measure cell viability/death after stress (e.g., dexamethasone treatment)

• Pathway analysis:

  • Consider using PCR arrays to identify affected signaling pathways (e.g., NFκB pathway)

This approach has been successfully implemented in mouse myogenic C2C12 and osteocyte-like MLO-Y4 cell lines, with observable phenotypic changes at days 3, 5, and 7 of myoblast differentiation .

What cellular models are most appropriate for studying bovine METTL21C function?

For studying bovine METTL21C function, the following cellular models are recommended:

For muscle-specific studies, both C2C12 cells and primary bovine myoblasts have proven valuable, as METTL21C function appears to be conserved across species in muscle tissue . For bone-related studies, MLO-Y4 cells have been successfully used to demonstrate METTL21C's role in osteocyte survival .

How does METTL21C affect myoblast differentiation?

METTL21C plays a crucial role in promoting myoblast differentiation, as evidenced by multiple studies:

• Expression pattern: METTL21C expression increases rapidly during myoblast differentiation, suggesting a developmental role .

• Knockdown effects: siRNA-mediated reduction of Mettl21c in C2C12 cells results in:

  • Reduced number of myocytes aligning for fusion at day 3 of differentiation

  • Fewer, shorter, and smaller myotubes at days 5 and 7

  • Significant reduction in fusion index (30.8 ± 1.6% in siRNA-treated cells vs. 37.0 ± 1.4% in controls)

  • Decreased myotube area (4324 ± 497.8 μm² in siRNA-treated vs. 9971 ± 471.7 μm² in controls)

• Overexpression effects: METTL21C overexpression in chicken myoblasts leads to:

  • Increased expression of myogenic markers MyoD and MyoG

  • Enhanced myoblast proliferation and differentiation

• Molecular mechanism: METTL21C appears to influence myoblast differentiation through:

  • Modulation of calcium homeostasis

  • Interaction with and methylation of chaperone proteins like Hsc70

  • Regulation of the NFκB signaling pathway

These findings collectively demonstrate that METTL21C functions as a positive regulator of myogenic differentiation across species.

How does METTL21C influence calcium homeostasis in muscle cells?

METTL21C plays a significant role in calcium homeostasis in muscle cells, which is critical for proper muscle function and development:

• Experimental evidence: siRNA-mediated knockdown of Mettl21c in C2C12 myotubes results in:

  • 16.1% decrease in amplitude peak Ca²⁺ response to caffeine (2.78 ± 0.03 vs. 3.31 ± 0.03 in controls)

  • 23.0% shorter relaxation phase of caffeine-induced calcium transients (85298 ± 395ms vs. 110802 ± 597ms in controls)

• Functional implications: These alterations suggest:

  • Reduced available Ca²⁺ for release from sarcoplasmic reticulum (SR)

  • Altered SR calcium handling machinery

  • Potential impact on excitation-contraction coupling

• Mechanistic hypotheses:

  • Direct modification of calcium handling proteins through METTL21C-mediated methylation

  • Indirect effects through methylation of chaperones that regulate calcium handling proteins

  • Secondary effect of impaired differentiation

For researchers investigating this aspect, calcium imaging techniques using fluorescent indicators like Fura-2 combined with caffeine stimulation provide valuable insights into METTL21C's role in muscle calcium dynamics .

What signaling pathways are modulated by METTL21C in muscle cells?

METTL21C primarily modulates the NFκB signaling pathway in muscle cells, as demonstrated by PCR array analysis following Mettl21c knockdown:

• Key affected genes in the NFκB pathway:

  • Birc3 (baculoviral IAP repeat-containing 3): An anti-apoptotic factor

  • Ccl5 (chemokine C-C motif ligand 5): Involved in myoblast migration and muscle regeneration

  • Tnf (tumor necrosis factor): A key inflammatory cytokine

• Functional relevance of NFκB signaling in muscle:

  • Critical for muscle homeostasis and regeneration

  • Regulates myoblast migration during development

  • Involved in inflammatory responses in muscle tissue

• Pathway connections:

  • Ccl5 is a downstream gene of NFκB and increases myoblast migratory activity

  • Ccl5 expression increases during muscle regeneration after injury

  • Birc3 mediates anti-cell death effects, potentially protecting developing myotubes

This modulation of NFκB signaling provides a potential mechanism through which METTL21C influences muscle development and function. Researchers investigating METTL21C should consider examining NFκB pathway components as key downstream effectors.

How does METTL21C affect osteocyte survival?

METTL21C plays a significant role in promoting osteocyte survival, particularly under stress conditions:

• Experimental evidence: In MLO-Y4 osteocyte-like cells, siRNA-mediated knockdown of Mettl21c followed by dexamethasone treatment (a known inducer of osteocyte apoptosis) resulted in significantly increased cell death compared to control cells .

• Methodological approach for studying this function:

  • Cell model: MLO-Y4 cells (osteocyte-like cell line)

  • Intervention: siRNA knockdown of Mettl21c

  • Stressor: Dexamethasone treatment (48 hours)

  • Readout: Cell viability assays (e.g., MTT, trypan blue exclusion)

• Potential mechanisms:

  • Regulation of NFκB signaling, which is known to influence cell survival

  • Methylation of proteins involved in cellular stress responses

  • Modulation of anti-apoptotic factors like Birc3

For researchers investigating METTL21C's role in bone biology, comparing its effects on different bone cell types (osteoblasts, osteocytes, osteoclasts) would provide valuable insights into its tissue-specific functions within the skeletal system.

What is the evidence supporting METTL21C as a pleiotropic gene for both bone and muscle?

Several lines of evidence support METTL21C as a pleiotropic gene influencing both bone and muscle:

• Genetic association: A bivariate genome-wide association study (GWAS) identified METTL21C as a suggestive pleiotropic locus (bivariate p = 2.3 ×10⁻⁷ for rs895999) associated with both bone and muscle traits .

• Functional evidence in muscle:

  • Essential for proper myoblast differentiation

  • Regulates calcium homeostasis in muscle cells

  • Highly expressed in skeletal muscle tissue

• Functional evidence in bone:

  • Promotes osteocyte survival under stress conditions

  • METTL21 family proteins methylate VCP chaperones, which are associated with Inclusion Body Myositis with Paget's Disease of bone

• Common signaling pathway: METTL21C modulates the NFκB signaling pathway, which is critical for both bone and muscle homeostasis .

• Evolutionary conservation: The role of METTL21C in muscle has been observed across multiple species (mouse, chicken, human), suggesting fundamental biological importance .

Researchers investigating the pleiotropic effects of METTL21C should consider dual-tissue experimental designs that examine bone and muscle phenotypes simultaneously, potentially using animal models with tissue-specific METTL21C manipulations.

How can I identify novel substrates of bovine METTL21C?

To identify novel substrates of bovine METTL21C, implement a multi-faceted approach:

• Unbiased biochemical screening:

  • Express and purify recombinant bovine METTL21C

  • Incubate with cellular lysates from bovine tissues (preferably muscle and bone)

  • Use [³H]-SAM as methyl donor

  • Identify methylated proteins by autoradiography followed by mass spectrometry

• Affinity purification coupled to mass spectrometry:

  • Generate tagged bovine METTL21C (e.g., FLAG, HA)

  • Express in bovine cells or tissues

  • Perform co-immunoprecipitation (Co-IP)

  • Identify interacting proteins by liquid chromatography-mass spectrometry (LC-MS/MS)

• Candidate approach based on structural similarities:

  • Analyze known substrates (AARS1, Hsc70) for common structural motifs

  • Search protein databases for bovine proteins with similar motifs

  • Test candidate proteins in vitro using methyltransferase assays

• Validation strategies:

  • Site-directed mutagenesis of potential methylation sites

  • In vitro methylation assays with purified substrates

  • In vivo confirmation using cell-based assays

This comprehensive approach has successfully identified AARS1 as a METTL21C substrate in previous research and should be adaptable to bovine-specific investigations .

What are the methodological challenges in studying species-specific differences in METTL21C function?

Investigating species-specific differences in METTL21C function presents several methodological challenges:

• Sequence and structural variation:

  • Amino acid differences between species may affect substrate specificity

  • Solution: Perform comparative sequence analysis and generate species-specific recombinant proteins

• Expression pattern differences:

  • Tissue distribution may vary between species

  • Solution: Conduct comprehensive expression profiling across tissues in multiple species

• Substrate availability:

  • Potential substrates may differ between species

  • Solution: Use species-matched substrates in enzymatic assays

• Functional readouts:

  • Different model systems may require different assays

  • Solution: Develop standardized assays applicable across species

• Cross-reactivity of research tools:

  • Antibodies may not recognize METTL21C across species

  • Solution: Validate antibodies for each species or use epitope tags

• Experimental design table for cross-species comparison:

Research on chicken METTL21C has provided valuable insights that may be applicable to bovine studies, particularly regarding its role in myoblast differentiation and interaction with Hsc70 .

How can CRISPR-Cas9 genome editing be utilized to study METTL21C function?

CRISPR-Cas9 genome editing offers powerful approaches for studying METTL21C function:

• Generation of knockout models:

  • Design sgRNAs targeting exons of bovine METTL21C

  • Create complete knockouts in relevant cell lines (bovine myoblasts, osteoblasts)

  • Analyze phenotypic consequences on differentiation, calcium signaling, and NFκB pathway

• Creation of catalytically inactive mutants:

  • Identify and mutate catalytic residues in the methyltransferase domain

  • Generate knock-in cell lines expressing catalytically dead METTL21C

  • Distinguish enzymatic from non-enzymatic functions

• Substrate validation:

  • Mutate methylation sites in putative substrates (e.g., AARS1-K943, Hsc70-K561)

  • Generate knock-in cell lines expressing unmethylatable substrate variants

  • Assess functional consequences of preventing specific methylation events

• Promoter studies:

  • Modify endogenous METTL21C promoter to understand regulation

  • Insert reporter genes to monitor expression patterns

  • Identify key regulatory elements controlling tissue-specific expression

• Tagging endogenous METTL21C:

  • Knock-in epitope tags or fluorescent proteins

  • Enable tracking of endogenous protein localization and interactions

  • Facilitate purification of native protein complexes

These CRISPR-based approaches would extend the siRNA knockdown studies that have already demonstrated METTL21C's importance in myoblast differentiation and osteocyte survival .

What are the potential contradictions in the current understanding of METTL21C substrate specificity?

Several contradictions exist in the current literature regarding METTL21C substrate specificity:

• VCP and HSPA8 as substrates:

  • Contradiction: Early reports claimed METTL21C methylates VCP and HSPA8

  • Conflicting evidence: More rigorous biochemical analyses found no evidence supporting these claims

  • Resolution approach: Direct comparative methylation assays with purified components and quantitative readouts

• METTL21 family substrate overlap:

  • Contradiction: Some studies suggest overlapping substrates between family members

  • Conflicting evidence: Research shows METTL21C specifically methylates AARS1, while other family members do not

  • Resolution approach: Structural studies to identify determinants of substrate specificity

• Species differences in substrates:

  • Contradiction: Hsc70 was identified as a substrate in chicken cells , but was not highlighted in mammalian studies

  • Resolution approach: Cross-species comparative analyses with standardized methodologies

• Methodological differences contributing to discrepancies:

  • In vitro vs. cellular assays

  • Overexpression vs. endogenous studies

  • Direct vs. indirect detection methods

To resolve these contradictions, researchers should:

• Perform side-by-side comparisons using identical experimental conditions

• Employ multiple complementary techniques to verify interactions and methylation

• Include appropriate positive and negative controls in all experiments

• Consider species-specific differences when interpreting results across model systems

The identification of AARS1 as a bona fide METTL21C substrate using unbiased biochemical approaches provides a strong foundation for resolving these contradictions .

How might METTL21C function contribute to musculoskeletal diseases?

METTL21C's dual role in muscle and bone biology suggests several potential contributions to musculoskeletal diseases:

• Muscle atrophy and sarcopenia:

  • METTL21C's critical role in myoblast differentiation suggests its dysregulation could impair muscle regeneration

  • Calcium homeostasis disruption caused by METTL21C deficiency may contribute to muscle weakness

  • NFκB pathway modulation by METTL21C could affect inflammatory processes in muscle atrophy

• Bone disorders:

  • METTL21C's promotion of osteocyte survival suggests potential involvement in osteoporosis

  • GWAS evidence links METTL21C to bone traits

  • METTL21 family proteins methylate VCP chaperones associated with Inclusion Body Myositis with Paget's Disease of bone

• Combined musculoskeletal conditions:

  • As a pleiotropic gene affecting both tissues, METTL21C may contribute to conditions with dual pathology

  • Relevant to conditions like sarcopenia with osteoporosis, a common comorbidity in aging

• Developmental disorders:

  • Given its role in myoblast differentiation, METTL21C mutations could potentially contribute to congenital myopathies

For researchers investigating these connections, examining METTL21C expression and mutation profiles in patient samples with various musculoskeletal diseases would be a valuable approach.

What experimental models are most suitable for studying METTL21C in a disease context?

Several experimental models are suitable for studying METTL21C in disease contexts:

For muscle-related studies, differentiation assays in C2C12 cells with METTL21C knockdown have successfully demonstrated its role in myogenesis . For bone studies, MLO-Y4 cells treated with dexamethasone provide a model for studying METTL21C in osteocyte survival under stress conditions .

Disease-specific approaches might include:

• For sarcopenia: Aged animal models with METTL21C manipulation

• For osteoporosis: Ovariectomized mice with METTL21C overexpression/knockdown

• For developmental disorders: CRISPR-engineered animal models with METTL21C mutations

These models would build upon the foundation established in basic research systems while extending findings toward clinical relevance.