METTL1 Human

What is METTL1 and what is its basic function in human cells?

METTL1 (methyltransferase like 1) is a protein-coding gene located on human chromosome 12q13 that encodes an RNA methyltransferase. It is a member of the Class I methyltransferase family with a highly conserved Rossmann-like fold consisting of seven β-sheet and six α-helices . The primary function of METTL1 is to introduce methyl groups to the N7 position of RNA molecules (particularly tRNAs) to form m7G (7-methylguanosine) modifications .

METTL1 functions in complex with its cofactor WDR4, forming a structure highly similar to the yeast tRNA m7G methyltransferase complex Trm8-Trm82 . The METTL1/WDR4 complex is responsible for catalyzing m7G methylation of various RNA types including mRNA, tRNA, rRNA, and miRNA . This modification plays essential roles in RNA stability, translation efficiency, and gene expression regulation .

What is the genomic structure and protein characteristics of human METTL1?

Human METTL1 is composed of 1,292 nucleotides encoding a 276 amino acid protein. The METTL1 gene contains seven exons and six introns, and its sequence shows similarity to the yeast open reading frame YDL201w . The gene has several synonyms including C12orf1, TRM8, TRMT8, and YDL201w .

METTL1 contains a conserved S-adenosylmethionine-binding motif that is critical for its methyltransferase activity . The protein structure includes the characteristic Rossmann-like fold common to Class I methyltransferases. The interaction between METTL1 and its cofactor WDR4 occurs specifically at the K143 domain of METTL1, which interacts with the R170 and E167 residues of WDR4, enhancing the enzymatic activity of the complex .

Of note, METTL1 activity can be regulated through phosphorylation; it is inactivated by phosphorylation , and specifically, the AKT signaling pathway can inhibit METTL1 enzyme activity through phosphorylation .

How does the METTL1/WDR4 complex function biochemically?

The METTL1/WDR4 complex functions as an m7G writer enzyme with WDR4 serving as a crucial cofactor. WDR4 is a 412 amino acid protein containing seven WD40 domains, located on human chromosome 21q22.3 . The interaction between these proteins occurs through specific regions:

• WDR4 consists of seven blades (B1-B7) forming a β-propeller structure with four seven β-sheet regions

• The B2-B5 region of WDR4 directly interacts with METTL1 and stabilizes the complex

• The R170 and E167 residues of WDR4 interact with the K143 domain of METTL1

Biochemically, WDR4 enhances the binding force between METTL1 and SAM (S-adenosylmethionine), which serves as the methyl donor, thereby promoting the methyltransferase activity of the complex . The METTL1/WDR4 complex primarily performs m7G modification at position 46 of the tRNA ring, which enhances mRNA translation by weakening ribosome suspension . Additionally, the complex mediates m7G labeling at position 1,639 of 18S rRNA and can perform m7G methylation modifications on miRNA and internal regions of mRNA .

What are the most effective methods for detecting and quantifying m7G modifications in RNA?

Multiple complementary approaches should be employed for comprehensive analysis of m7G modifications:

• HPLC-MS (High-Performance Liquid Chromatography-Mass Spectrometry): This is a gold standard method for quantifying m7G levels in tRNA. As demonstrated in recent studies, HPLC-MS can confirm changes in tRNA m7G levels following METTL1 overexpression or knockout . This technique provides absolute quantification of modification levels.

• RNA sequencing approaches:

  • Standard RNA-seq can be used to identify METTL1-regulated genes by comparing expression profiles between METTL1 knockdown and control samples .

  • Specialized sequencing techniques that specifically capture m7G-modified RNA can provide genome-wide maps of m7G modifications.

• Immunoprecipitation-based techniques: Antibodies specific to m7G can be used to enrich modified RNAs, followed by sequencing or other detection methods.

• Mutational analysis: Creating catalytically inactive mutants (such as the AFPA-mutant of METTL1) can serve as important controls in functional studies .

For optimal accuracy, researchers should combine these approaches to validate findings across multiple methodologies.

What experimental strategies are best for investigating METTL1 function in cellular models?

To comprehensively investigate METTL1 function in cellular models, researchers should consider the following approaches:

• Genetic manipulation strategies:

  • Knockdown using siRNA/shRNA: Effective for temporary reduction of METTL1 expression

  • CRISPR-Cas9 knockout: For complete elimination of METTL1 function

  • Overexpression systems: Using intact METTL1 compared to catalytically inactive mutants (e.g., AFPA-mutant)

  • Conditional systems: For temporal control of expression

• Functional readouts:

  • Proliferation assays: EdU incorporation can measure cell proliferation rates (e.g., cells overexpressing METTL1 showed approximately 35% EdU-positive cells compared to 22% in control cells)

  • Senescence assays: SA-β-gal staining to detect senescent cells

  • Protein level analysis: Western blotting for senescence markers like LMNB1, EZH2, and p16

  • RNA translation efficiency: Ribosome profiling to detect stalling at specific codons

• Pathway analysis:

  • GO and KEGG enrichment analysis of differentially expressed genes following METTL1 manipulation

  • Protein interaction studies to identify METTL1-associated proteins

• Molecular modeling:

  • Crystal structure modeling of the METTL1/WDR4 complex can provide insights into functional domains and potential drug targeting sites

How can researchers effectively design experiments to study the role of METTL1 in animal models?

When designing animal experiments to study METTL1, researchers should consider the following approaches:

• Genetic model systems:

  • Conditional knockout (cKO) models: Allow tissue-specific or temporally controlled deletion of Mettl1

  • Conditional knock-in (cKI) models: For controlled overexpression studies

  • These models can be created by crossing Mettl1-floxed mice with appropriate Cre-expressing lines

• Phenotypic analyses:

  • Lifespan studies: METTL1 knockdown has been shown to significantly shorten the lifespan of mice

  • Aging markers: Assessment of premature aging phenotypes in various tissues

  • Tissue damage analysis: Particularly following stressors like chemotherapy agents

• Mechanistic studies:

  • Tissue-specific RNA methylation analysis: Using techniques like HPLC-MS to quantify m7G levels in different tissues

  • Ribosome profiling: To identify tissue-specific translation effects of METTL1 deficiency

  • Stress response pathway analysis: Examination of ribotoxic stress response (RSR) and integrated stress response (ISR) activation

• Rescue experiments:

  • Testing whether overexpression of downstream effectors (e.g., eEF1A protein) can mitigate phenotypes caused by METTL1 deficiency

  • Administration of compounds that target the stress pathways activated by METTL1 deficiency

How does METTL1-mediated m7G methylation regulate pluripotency in human stem cells?

METTL1-mediated m7G methylation plays a crucial role in maintaining pluripotency in human stem cells through several mechanisms:

• Translational regulation of pluripotency factors: METTL1 influences the translation process through methylation of various RNA species, including mRNA, tRNA, and miRNA . This translational control is particularly important for pluripotency-associated genes.

• Impact on stem cell self-renewal: Research has demonstrated that METTL1-mediated m7G tRNA modification regulates the self-renewal and pluripotency of stem cells . The METTL1/WDR4 complex influences tRNA function, ribosome suspension, and mRNA translation in embryonic stem cells .

• Neural lineage differentiation regulation: Studies in mouse embryonic stem cells (mESCs) have shown that METTL1 is involved in regulating neural lineage differentiation . This suggests a role in directing cell fate decisions during development.

• Cell cycle regulation: METTL1 is particularly sensitive to the translation of cell cycle genes , which are critical for the rapid proliferation characteristic of pluripotent stem cells.

• Codon-specific translation effects: The m7G modification at position 46 of tRNA enhances mRNA translation efficiency by preventing ribosome stalling at specific codons . This mechanism allows for the selective translation of mRNAs enriched in particular codons, potentially including those coding for pluripotency factors.

These findings highlight METTL1 as a critical regulator of stem cell fate through its effects on RNA methylation and subsequent translational control.

What techniques are most effective for analyzing METTL1's impact on stem cell differentiation?

To comprehensively analyze METTL1's impact on stem cell differentiation, researchers should employ a multi-faceted approach:

These approaches should be integrated to provide a comprehensive understanding of how METTL1-mediated m7G modifications influence stem cell fate decisions and differentiation pathways.

What is the role of METTL1 in cancer progression and how can it be targeted therapeutically?

METTL1 plays significant roles in cancer progression through multiple mechanisms:

• Cancer cell growth and proliferation:

  • Upregulated expression of the METTL1/WDR4 complex has been observed in various cancers and is associated with malignancy and poor survival

  • Loss of METTL1 and WDR4 impairs cancer cell growth, tumorigenesis, and malignant transformation

  • METTL1 knockdown reduces downstream signaling activity in the EGF/EGFR and VEGFA/VEGFR1 pathways, inhibiting proliferation

• Cell cycle regulation:

  • METTL1 knockdown inhibits Cyclin A2 (CCNA2) translation, leading to cell cycle arrest

  • During the cell cycle, METTL1 protects specific mRNAs from degradation through tRNA methylation

• Invasion and metastasis:

  • METTL1-mediated m7G tRNA modifications selectively modulate the translation of key genes in the epithelial to mesenchymal transition (EMT) process

  • The METTL1-m7G-SLUG/SNAIL axis is important for preventing hepatocellular carcinoma (HCC) metastasis

• Stress response and chemoresistance:

  • m7G in tRNA protects cancer cells from stress-induced cutting and processing of 5′tRNA fragments

  • Lack of m7G methylation activates stress response pathways, making cancer cells more susceptible to stress

Therapeutic targeting approaches:

• Direct METTL1 inhibition:

  • Development of small molecule inhibitors targeting the METTL1/WDR4 complex

  • The crystal structure model of human METTL1 with WDR4 provides a foundation for rational drug design

• Combination therapies:

  • Targeting METTL1 may increase cancer sensitivity to chemotherapy

  • Combining METTL1 inhibitors with stress-inducing treatments could be particularly effective

• Pathway-specific approaches:

  • Targeting the PTEN/AKT signaling pathway, which interacts with METTL1's carcinogenic activity

  • Focusing on the METTL1-m7G-SLUG/SNAIL axis to prevent metastasis

The METTL1/WDR4 complex is increasingly recognized as a potential target for cancer treatment , particularly for cancers where overexpression has been documented.

How does METTL1 expression correlate with clinical outcomes in various cancer types?

METTL1 expression has significant correlations with clinical outcomes across different cancer types:

• General cancer correlation patterns:

  • Upregulated expression of the METTL1/WDR4 complex is observed in various cancers

  • Higher expression levels correlate with increased malignancy and poor survival outcomes

  • Integrated genomic, transcriptomic, proteomic, and clinicopathological analyses of METTL1 in large cohorts of primary tumors and cell lines have identified it as a top candidate gene with significant clinical implications

• Hepatocellular carcinoma (HCC):

  • METTL1 plays multiple roles in HCC progression including:

    • Promoting tumorigenesis through activation of EGF/EGFR and VEGFA/VEGFR1 signaling pathways

    • Cell cycle regulation through control of CCNA2 translation

    • Enhancing invasion and migration via the METTL1-m7G-SLUG/SNAIL axis

  • METTL1 knockdown reduces m7G modification of tRNA in HCC cells, affecting target mRNA translation with high frequency of m7G-related codons

  • METTL1 exhibits carcinogenic activity through the PTEN/AKT signaling pathway in HCC

• Acute myeloid leukemia:

  • METTL1 plays a role in the growth of acute myeloid leukemia cells

  • The regulation of tRNA m7G methylation affects leukemia cell survival under stress conditions

• Correlation with treatment response:

  • Loss of METTL1 makes cancer cells more susceptible to stress, suggesting potential synergy with chemotherapy

  • Targeting the METTL1-m7G axis may prevent HCC metastasis particularly after radiofrequency thermal ablation therapy

These findings suggest that METTL1 expression and activity could serve as both prognostic markers and potential therapeutic targets across multiple cancer types.

How does METTL1 deficiency contribute to cellular senescence and aging?

METTL1 deficiency contributes to cellular senescence and aging through several interconnected mechanisms:

• Translational defects due to tRNA hypomethylation:

  • METTL1 and WDR4 are downregulated during cell senescence and aging at both transcriptional and protein levels

  • This downregulation results in a subset of tRNAs being targeted for rapid tRNA decay (RTD) degradation due to m7G46 hypomethylation

  • The loss of properly modified tRNAs impairs efficient translation of specific mRNAs

• Ribosome stalling and stress response activation:

  • METTL1 deficiency causes ribosomes to stall at certain codons, impeding the translation of mRNAs essential in critical pathways including:

    • Wnt signaling pathway

    • Ribosome biogenesis pathway

  • Chronic ribosome stalling triggers two major stress responses:

    • Ribotoxic stress response (RSR)

    • Integrative stress response (ISR)

  • These stress responses induce the senescence-associated secretory phenotype (SASP)

• Cell cycle and proliferation effects:

  • Knockdown of METTL1 drives proliferating cells into senescence

  • EdU incorporation assays show significantly reduced proliferation in METTL1-deficient cells compared to cells with intact METTL1 (approximately 22% vs. 35% EdU-positive cells)

  • SA-β-gal staining confirms increased senescence in METTL1-deficient cells

• Lifespan and aging impact:

  • METTL1 deficiency significantly shortens the lifespan of mice in vivo

  • Conditional knockout models demonstrate premature aging phenotypes

These findings establish METTL1-mediated tRNA m7G modification as essential for preventing premature senescence and aging by enabling efficient mRNA translation and preventing detrimental stress responses.

Can METTL1 overexpression mitigate aging-related phenotypes, and what experimental approaches best demonstrate this?

METTL1 overexpression shows promising results in mitigating aging-related phenotypes through several mechanisms:

• Cellular senescence delay:

  • Overexpression of intact METTL1 (but not the catalytically inactive AFPA-mutant) delays cell senescence

  • Cells overexpressing METTL1 maintain higher proliferation rates, with approximately 35% EdU-positive cells compared to 22% in control cells

  • SA-β-gal staining demonstrates significantly decreased populations of senescent cells in METTL1-overexpressing cultures

• Restoration of senescence markers:

  • METTL1 overexpression partially restores levels of senescence-associated proteins:

    • LMNB1 (Lamin B1, which decreases during senescence)

    • EZH2 (which also decreases during senescence)

    • Reduces p16 levels (a key senescence marker)

• Protection against tissue damage:

  • METTL1 overexpression mitigates tissue damage induced by chemotherapy agents

  • This suggests broader protective effects against various stressors that accelerate aging

Optimal experimental approaches to demonstrate these effects include:

• In vitro approaches:

  • Stable cell lines with doxycycline-inducible METTL1 expression

  • Comparison between wild-type METTL1 and catalytically inactive mutants (e.g., AFPA-mutant)

  • Replicative senescence models (serial passaging)

  • Stress-induced senescence models (oxidative stress, DNA damage)

  • Comprehensive senescence marker analysis (SA-β-gal, p16, p21, LMNB1, SASP factors)

• In vivo approaches:

  • Conditional transgenic models with tissue-specific METTL1 overexpression

  • Lifespan studies in these models

  • Healthspan assessments (physical performance, metabolic parameters)

  • Challenge models with aging accelerators (chemotherapy, radiation)

  • Age-related pathology assessment in multiple organ systems

• Mechanistic validation:

  • tRNA methylation status monitoring via HPLC-MS

  • Ribosome profiling to confirm improved translation efficiency

  • Assessment of stress response pathway activation (RSR and ISR)

  • Rescue experiments comparing METTL1 overexpression to known mediators like eEF1A

These approaches collectively provide a comprehensive evaluation of METTL1's potential as an intervention target for aging and senescence-related conditions.

Advanced Research Questions in METTL1 Biology

Determinants of substrate preference:

• Sequence context: Specific nucleotide sequences surrounding the target guanosine likely influence METTL1 recognition

• RNA structural motifs: Secondary and tertiary structural elements may create recognition sites for the METTL1/WDR4 complex

• Cofactor interactions: The interaction with WDR4 enhances METTL1's binding to SAM and may influence substrate recognition

• Cellular compartmentalization: Localization of METTL1 may determine access to different RNA populations

• Competition with other modification enzymes: Other RNA modification pathways may compete for the same substrates

Further research using techniques such as CLIP-seq (cross-linking immunoprecipitation followed by sequencing) would be valuable for comprehensively mapping METTL1 binding sites across the transcriptome and identifying consensus recognition motifs.

What are the mechanisms of cross-talk between METTL1-mediated m7G modification and other RNA modifications?

The cross-talk between METTL1-mediated m7G modification and other RNA modifications represents a complex regulatory network:

• Interaction with m6A pathways:

  • RNA m6A (N6-methyladenosine) modification has been observed during embryogenesis and is involved in stem cell development

  • Both m7G and m6A can influence RNA stability and translation, suggesting potential synergistic or antagonistic effects

  • The precise mechanisms of cross-talk between these pathways in regulating stem cell fate requires further investigation

• Cooperative modification patterns:

  • Multiple modifications on the same RNA molecule can create "modification codes" that determine RNA fate

  • The presence of one modification may enhance or inhibit the addition of others

  • In tRNAs, which contain numerous modifications, m7G at position 46 may influence the addition or function of other modifications

• Shared regulatory mechanisms:

  • METTL1 enzyme activity is regulated by phosphorylation via the AKT pathway

  • This pathway may simultaneously regulate other RNA modification enzymes, creating coordinated responses

  • The mTOR signaling pathway also regulates METTL1 enzyme activity , and mTOR is known to influence multiple RNA modification pathways

• Competition for substrate RNAs:

  • Different RNA modification enzymes may compete for the same RNA substrates

  • The balance between various modifications may determine the ultimate fate of the RNA molecule

• Modification interplay in stress response:

  • METTL1-mediated m7G in tRNA protects from stress-induced cutting and processing of 5′tRNA fragments

  • Other stress-responsive RNA modifications may work in concert with or opposition to this protective function

• Epigenetic regulation of modification enzymes:

  • The P300/SP1 complex modulates METTL1 activity

  • Similar epigenetic regulatory mechanisms may coordinate the expression of multiple RNA modification enzymes

Understanding these cross-talk mechanisms requires integrated approaches combining transcriptome-wide mapping of multiple modifications, functional studies of enzymes and their regulators, and computational modeling of modification networks.

How do evolutionary changes in METTL1 relate to species-specific differences in development and disease susceptibility?

Evolutionary analysis of METTL1 provides important insights into its conserved functions and species-specific adaptations:

• Evolutionary conservation of core function:

  • METTL1 is similar in sequence to the S. cerevisiae YDL201w gene , indicating ancient evolutionary origins

  • The METTL1/WDR4 complex is highly similar to the yeast tRNA m7G methyltransferase complex Trm8-Trm82

  • This conservation suggests a fundamental role in cellular processes that has been maintained throughout eukaryotic evolution

• Structural conservation and divergence:

  • The core methyltransferase domain with the Rossmann-like fold is highly conserved across species

  • The S-adenosylmethionine-binding motif is preserved as a critical functional element

  • Species-specific variations in protein sequence may influence:

    • Substrate specificity

    • Regulatory mechanisms

    • Protein-protein interactions

• Developmental implications:

  • METTL1 and WDR4 mutations in humans can lead to primordial dwarfism and brain malformation

  • Evolutionary changes in METTL1 regulation or activity may contribute to species-specific:

    • Brain development patterns

    • Growth trajectories

    • Stem cell regulation

• Disease susceptibility differences:

  • Species-specific METTL1 functions may influence comparative cancer biology

  • Different regulation of METTL1 across species may affect:

    • Aging processes

    • Stress responses

    • Susceptibility to neurodevelopmental disorders

• Comparative regulatory mechanisms:

  • While core enzymatic functions are conserved, regulatory mechanisms controlling METTL1 expression and activity likely vary across species

  • These differences may contribute to species-specific developmental timing, stress responses, and disease susceptibilities

Research approaches to explore these evolutionary aspects should include:

• Comparative genomic analysis across diverse species

• Functional complementation studies

• Creation of humanized animal models

• Analysis of species-specific METTL1 interactomes

• Detailed structural comparison of METTL1/WDR4 complexes across species

What are the current therapeutic approaches targeting METTL1 in development, and what challenges remain?

Current therapeutic approaches targeting METTL1 are in early stages of development but show promising potential:

• Direct inhibition strategies:

  • Small molecule inhibitors targeting the METTL1/WDR4 complex are being developed

  • Crystal structure modeling of human METTL1 with its cofactor WDR4 provides a foundation for rational drug design

  • Molecular modeling and simulation approaches help identify potential binding sites for inhibitors

• Combination therapy approaches:

  • METTL1 inhibition may increase cancer sensitivity to chemotherapy

  • Targeting METTL1 alongside conventional cancer treatments could enhance efficacy

  • The METTL1-m7G-SLUG/SNAIL axis is being explored as a target to prevent HCC metastasis after radiofrequency thermal ablation therapy

• Pathway-based interventions:

  • Targeting the interaction between METTL1 and regulatory pathways like P300/SP1

  • Modulating mTOR signaling, which regulates METTL1 enzyme activity

  • Focusing on stress response pathways activated by METTL1 deficiency

Major challenges in METTL1-targeted therapeutics include:

• Specificity concerns:

  • Ensuring inhibitors are specific to METTL1/WDR4 without affecting other methyltransferases

  • Avoiding disruption of essential cellular processes in normal cells

• Delivery challenges:

  • Developing effective delivery systems to target METTL1 inhibitors to specific tissues

  • Overcoming barriers to reach intracellular targets

• Context-dependent effects:

  • METTL1 inhibition may be beneficial in cancer but detrimental for aging-related conditions

  • Tissue-specific and disease-specific approaches are needed

• Biomarker development:

  • Identifying reliable biomarkers to predict response to METTL1-targeted therapies

  • Developing methods to monitor m7G levels as pharmacodynamic markers

• Resistance mechanisms:

  • Understanding potential compensatory pathways that may emerge following METTL1 inhibition

  • Developing strategies to prevent or overcome resistance

While the METTL1/WDR4 complex is increasingly recognized as a potential target for cancer treatment , significant research is still needed to translate these findings into clinically viable therapeutic approaches.

How can METTL1 dysfunction contribute to neurological disorders, and what are the therapeutic implications?

METTL1 dysfunction has significant implications for neurological disorders through several mechanisms:

• Neurodevelopmental impacts:

  • Mutations in the human m7G methyltransferase complex METTL1/WDR4 can lead to brain malformation

  • The METTL1/WDR4 complex influences genes associated with brain abnormalities in mouse embryonic stem cells

  • METTL1 plays a role in neural lineage differentiation , suggesting impacts on neurogenesis

• Translational regulation in neurons:

  • METTL1-mediated m7G tRNA modification regulates mRNA translation

  • Neurons rely heavily on precise translational control, particularly in:

    • Synaptic plasticity

    • Dendritic protein synthesis

    • Response to neuronal activity

  • Dysregulation of this process could impact neuronal function and connectivity

• Stress response in neural cells:

  • METTL1 deficiency triggers ribotoxic stress response (RSR) and integrative stress response (ISR)

  • Neural cells are particularly vulnerable to protein folding stress and translational dysregulation

  • Chronic stress pathway activation contributes to neurodegeneration

• Aging-related neurological decline:

  • METTL1 deficiency drives cellular senescence

  • Cellular senescence in the brain contributes to aging-related cognitive decline

  • The senescence-associated secretory phenotype (SASP) induced by METTL1 deficiency can promote neuroinflammation

Therapeutic implications and approaches:

• Developmental disorders:

  • Early diagnosis of METTL1/WDR4 mutations for intervention in neurodevelopmental disorders

  • Potential gene therapy approaches to restore proper m7G methylation during development

• Neurodegenerative diseases:

  • METTL1 overexpression strategies may delay neural senescence

  • Targeting downstream stress pathways activated by METTL1 deficiency

  • Restoring translation efficiency by modulating eEF1A levels, which can mitigate senescence phenotypes caused by METTL1 deficiency

• Stroke and neural injury:

  • METTL1 overexpression may protect against tissue damage , potentially including neural tissue

  • Therapeutic interventions targeting the translation stress response following neural injury

• Biomarker development:

  • m7G levels in accessible biofluids as potential biomarkers for neurological disease progression

  • Monitoring METTL1 expression or activity as a diagnostic tool