GO:0016428 tRNA (cytidine-N5)-methyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016428 describes the enzymatic activity that transfers a methyl group from S-adenosyl-L-methionine to the N5 position of cytidine in tRNA, producing 5-methylcytidine.
• The reaction can occur at several tRNA cytidine residues, including positions 34, 40, 48, and 49, and influences tRNA stability, decoding, and translation.
• tRNA methylation is part of a broader RNA modification network that includes m7G, m1A, and m5C, and dysregulation of these marks is linked to cancer, autoimmunity, and aging [1,2,4,5,7,8].
• Key enzymes and cofactors include METTL1-WDR4 for m7G, TRMT6/TRMT61A for m1A, and the DNMT family for cytosine methylation, illustrating conserved methyltransferase mechanisms [1,3,4,7].
• Loss- or gain-of-function studies using CRISPR knockout, point mutation, and knock-in models are essential to dissect how tRNA cytidine methylation affects cellular phenotypes [5,6,8].
• EDITGENE provides end-to-end CRISPR services, including knockout, point mutation, knock-in, overexpression, library screening, and bioinformatics, to accelerate tRNA modification research [5,6,8].
Description
GO:0016428, tRNA (cytidine-N5)-methyltransferase activity, is a molecular function that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the N5 position of cytidine within tRNA, generating 5-methylcytidine and S-adenosyl-L-homocysteine. This modification can occur on several cytidine residues, including positions 34, 40, 48, and 49, and represents one of the many post-transcriptional RNA modifications that fine-tune tRNA function. Understanding this activity is important because tRNA modifications influence translation fidelity, tRNA stability, and cellular stress responses, and their dysregulation has been implicated in cancer, metabolic reprogramming, and autoimmune conditions [1,2,4,5,7,8]. Researchers studying GO:0016428 often focus on the enzymes that catalyze the reaction, the structural basis of substrate recognition, and the downstream biological consequences of altered methylation [1,4,6].
tRNA (cytidine-N5)-methyltransferase activity At A Glance
| GO ID | GO:0016428 |
|---|---|
| GO term | tRNA (cytidine-N5)-methyltransferase activity |
| Ontology | molecular_function |
| Synonym | S-adenosyl-L-methionine:tRNA (cytosine-5-)-methyltransferase activity; transfer ribonucleate cytosine 5-methyltransferase activity; transfer RNA cytosine 5-methyltransferase activity; tRNA (cytidine-5-)-methyltransferase activity; tRNA (cytosine-5-)-methyltransferase activity |
| Major function | Catalyzes the methylation of cytidine in tRNA using S-adenosyl-L-methionine, producing 5-methylcytidine in tRNA, S-adenosyl-L-homocysteine, and H+ |
| Substrate | Cytidine in tRNA and S-adenosyl-L-methionine |
| Products | 5-methylcytidine in tRNA, S-adenosyl-L-homocysteine, and H+ |
| Modification sites | Cytidine(34), cytidine(40), cytidine(48), and cytidine(49) in tRNA |
| Related modifications | m7G, m1A, and other tRNA methylation marks [1,4,7] |
What Is GO:0016428?
In my own words, GO:0016428 describes the catalytic activity of an enzyme that uses S-adenosyl-L-methionine as a methyl donor to add a methyl group to the N5 nitrogen of a cytidine nucleotide in a tRNA molecule. The reaction yields a 5-methylcytidine in tRNA, S-adenosyl-L-homocysteine, and a proton. This activity can target multiple cytidine positions within tRNA, such as cytidine(34), cytidine(40), cytidine(48), and cytidine(49), and is therefore not restricted to a single site. The modification is part of the broader landscape of tRNA methylation that includes m7G, m1A, and other marks, and it contributes to the structural and functional maturation of tRNA.
Why Is tRNA (cytidine-N5)-methyltransferase activity Important in Cell Biology?
GO:0016428 is important because tRNA cytidine methylation is a conserved post-transcriptional modification that affects tRNA structure, stability, and decoding capacity, and its dysregulation has been linked to human diseases including cancer, autoimmunity, and aging [1,2,4,5,7,8]. Studying this activity helps researchers understand how cells maintain translation fidelity under stress and how metabolic and immune pathways are rewired in disease states [2,5,8].
• tRNA cytidine methylation contributes to tRNA stability and proper folding, influencing translation efficiency.
• Altered tRNA methylation is observed in cancer, where it can drive metabolic plasticity and tumorigenesis [2,7].
• Dysregulation of tRNA modifications, including m7G and m1A, is linked to autoimmune responses and B-cell dysfunction.
• Loss of tRNA methylation can induce senescence and aging phenotypes in cellular and animal models.
• Methyltransferase enzymes often have modification-independent roles, complicating the interpretation of knockout studies.
• Structural studies of methyltransferase complexes provide a framework for understanding substrate recognition and catalysis [1,4].
• tRNA modification pathways are emerging as potential therapeutic targets in oncology and immunology [2,7,8].
• CRISPR-based models enable precise dissection of enzyme function and modification site specificity [5,6,8].
What Happens During tRNA (cytidine-N5)-methyltransferase activity?
Substrate recognition and binding
In simple terms: The enzyme first finds and grabs the correct tRNA and the methyl donor.
The methyltransferase recognizes specific structural features of tRNA, including the anticodon loop and other regions, to select target cytidine residues such as positions 34, 40, 48, and 49. Binding of S-adenosyl-L-methionine positions the methyl group for transfer, and structural studies of related methyltransferases, such as METTL1-WDR4, reveal how substrate specificity is achieved [1,4].
Methyl group transfer
In simple terms: The enzyme moves a methyl group from SAM onto the cytidine ring.
The catalytic step involves nucleophilic attack by the N5 nitrogen of cytidine on the methyl group of S-adenosyl-L-methionine, resulting in 5-methylcytidine and S-adenosyl-L-homocysteine. This reaction is analogous to other cytosine methyltransferases, such as the DNA methyltransferase family, which use a conserved catalytic mechanism.
Product release and tRNA maturation
In simple terms: After modification, the tRNA is released and can function in translation.
Following methyl transfer, the modified tRNA is released, and the 5-methylcytidine mark can influence tRNA stability, folding, and interaction with the translation machinery. The modification may also affect codon-anticodon pairing and ribosomal decoding, as suggested by studies of other tRNA modifications [1,4].
Integration with other tRNA modifications
In simple terms: Cytidine methylation works together with other tRNA marks.
tRNA cytidine methylation is part of a broader modification network that includes m7G, m1A, and other marks, and these modifications can influence each other's deposition and function [1,4,7]. For example, METTL1-mediated m7G modification and TRMT6/TRMT61A-mediated m1A modification are critical for tRNA function and are linked to disease [1,4,7].
Key Genes Involved in GO:0016428 tRNA (cytidine-N5)-methyltransferase activity
The following genes and proteins are directly or functionally associated with tRNA methylation and related RNA modification pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| METTL1 | Catalytic subunit of the m7G tRNA methyltransferase complex | Structural and functional studies of tRNA methylation; linked to cancer and autoimmunity [1,4,8] |
| WDR4 | Essential cofactor for METTL1-mediated m7G modification | Required for METTL1 stability and activity; mutations affect tRNA modification [1,4] |
| TRMT6 | Subunit of the m1A tRNA methyltransferase complex | tRNA m1A modification; implicated in liver tumorigenesis |
| TRMT61A | Catalytic subunit of the m1A tRNA methyltransferase complex | tRNA m1A modification; regulates cholesterol metabolism in liver cancer |
| DNMT1 | DNA methyltransferase | Model for cytosine methylation mechanisms; conserved catalytic motifs |
| DNMT3A | DNA methyltransferase | De novo methylation; provides mechanistic parallels to RNA cytosine methylation |
| DNMT3B | DNA methyltransferase | De novo methylation; insights into methyltransferase regulation |
| NSUN2 | RNA cytosine methyltransferase (m5C) | Catalyzes m5C in tRNA and other RNAs; related to GO:0016428 activity |
| NSUN6 | RNA cytosine methyltransferase (m5C) | Targets specific tRNA positions; potential overlap with cytidine methylation pathways |
| ALKBH1 | tRNA demethylase | Reverses m1A and other modifications; balances methylation status |
| FTO | RNA demethylase | Regulates m6A and other modifications; impacts tRNA modification crosstalk |
| METTL3 | m6A methyltransferase | Model for RNA methylation; informs general methyltransferase mechanisms |
| WTAP | m6A methyltransferase complex subunit | Regulates METTL3 activity; parallels to tRNA methyltransferase complexes |
| TRMT112 | Partner of NSUN2 and other methyltransferases | Stabilizes and activates tRNA methyltransferases |
| YBX1 | RNA-binding protein | Facilitates tRNA modification and stability; interacts with methyltransferases |
| ELP1 | tRNA modification complex component | Affects tRNA wobble modifications; related to translation fidelity |
| ELP3 | tRNA modification enzyme | Catalyzes carboxymethylation; crosstalk with methylation pathways |
How Is tRNA (cytidine-N5)-methyltransferase activity Regulated?
The activity of tRNA (cytidine-N5)-methyltransferase is regulated at multiple levels, including enzyme expression, complex assembly with cofactors such as WDR4, and availability of the methyl donor S-adenosyl-L-methionine [1,4]. Cellular stress, metabolic state, and oncogenic signaling can influence tRNA modification patterns, as shown for m7G and m1A pathways [2,5,7,8]. For example, mitochondrial RNA modifications shape metabolic plasticity during metastasis, indicating that nutrient and metabolic cues regulate RNA methylation. Additionally, perturbation of METTL1-mediated m7G modification induces senescence and aging, suggesting that cellular stress pathways modulate methyltransferase activity.
tRNA (cytidine-N5)-methyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| METTL1 | Cancer, autoimmunity, aging | Knockout and point-mutation cell lines; mouse models [1,4,5,8] |
| TRMT6/TRMT61A | Liver cancer, cholesterol metabolism | Knockout hepatoma cells; xenograft models |
| NSUN2 | Cancer, neurodevelopmental disorders | Knockout and overexpression cell models |
| FTO | Metabolic disorders, cancer | Knockout and point-mutation models |
| ALKBH1 | Cancer, neurological disease | Knockout and knock-in models |
Cancer and metabolic reprogramming
Dysregulation of tRNA modifications, including m7G and m1A, is linked to cancer progression and metabolic reprogramming. METTL1-mediated m7G modification supports oncogenic transformation, and TRMT6/TRMT61A-mediated m1A drives liver tumorigenesis by regulating cholesterol metabolism [1,4,7]. Mitochondrial RNA modifications also shape metabolic plasticity in metastasis, highlighting the broad impact of RNA methylation on cancer metabolism.
Autoimmunity and immune dysfunction
Aberrant METTL1-mediated tRNA m7G modification alters B-cell responses in systemic autoimmunity, demonstrating that tRNA methylation pathways are critical for immune homeostasis. This suggests that cytidine methylation and related modifications may contribute to autoimmune pathogenesis.
Aging and senescence
Perturbation of METTL1-mediated tRNA m7G modification induces senescence and aging, indicating that loss of tRNA methylation can accelerate aging phenotypes. This connects tRNA modification enzymes to cellular longevity and stress responses.
Methyltransferase-independent roles
A methyltransferase-independent role for METTL1 in tRNA aminoacylation and oncogenic transformation has been reported, underscoring the complexity of interpreting disease associations when enzymes have multiple functions.
From tRNA (cytidine-N5)-methyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of tRNA cytidine methyltransferase affect translation? | CRISPR knockout cell lines followed by polysome profiling |
| Which cytidine residues are targeted? | Point-mutation knock-in of tRNA genes or enzyme active site |
| Can a disease-associated mutation alter enzyme activity? | Knock-in of patient-derived mutations |
| Does overexpression drive oncogenic transformation? | Stable overexpression cell lines and xenografts |
| What proteins interact with the methyltransferase? | Tagged knock-in for affinity purification and proteomics [1,4] |
| How does the modification change during stress? | Knockout and rescue models under stress conditions [2,5] |
How to Study the tRNA (cytidine-N5)-methyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Transcript abundance and splicing | Global gene expression changes upon knockout |
| m5C RNA IP | Cytosine methylation sites | Mapping tRNA modification sites |
| Ribo-seq | Translation efficiency and ribosome occupancy | Codon-specific effects of tRNA modification [1,4] |
| Polysome profiling | Active translation complexes | Assessing translation defects in knockout cells |
| Mass spectrometry | Protein interactions and modifications | Identifying methyltransferase complexes [1,4] |
| Cryo-EM | 3D structures of macromolecules | Structural basis of substrate recognition [1,4] |
| CRISPR screening | Gene essentiality and modifiers | Discovering regulators of tRNA methylation [6,8] |
RNA sequencing and modification mapping
RNA-seq and specialized modification mapping techniques, such as m5C RNA immunoprecipitation, can identify tRNA cytidine methylation sites and quantify changes upon enzyme perturbation. These methods are essential for linking GO:0016428 activity to specific tRNA substrates.
Ribosome profiling and translation assays
Ribo-seq and polysome profiling measure translation efficiency and codon-specific effects caused by altered tRNA methylation [1,4]. Such approaches reveal how modifications influence decoding and protein synthesis.
Proteomics and interactomics
Affinity purification coupled with mass spectrometry identifies protein complexes containing tRNA methyltransferases, such as METTL1-WDR4, and reveals modification-independent interactions [1,4,6].
Structural biology and imaging
Cryo-EM and X-ray crystallography provide atomic-level views of methyltransferase complexes, while fluorescence imaging can track tRNA localization and modification dynamics [1,4].
How CRISPR Can Be Used to Study GO:0016428 tRNA (cytidine-N5)-methyltransferase activity
Knockout
CRISPR knockout of tRNA methyltransferase genes, such as METTL1 or NSUN2, allows researchers to assess loss-of-function phenotypes, including changes in tRNA modification, translation, and cell viability [5,6,8]. Knockout models are foundational for establishing causality between GO:0016428 activity and cellular processes.
Point Mutation
Point mutations in the catalytic domain or substrate-binding pocket can dissect enzymatic activity from other functions. For example, methyltransferase-dead mutants of METTL1 reveal modification-independent roles in tRNA aminoacylation and transformation.
Knock-in
Knock-in of disease-associated mutations or epitope tags enables precise modeling of patient variants and facilitates protein interaction studies [1,4,8]. Tagged knock-in lines are valuable for affinity purification and imaging.
Overexpression
Overexpression of wild-type or mutant methyltransferases can drive oncogenic phenotypes and reveal gain-of-function mechanisms, as shown for METTL1 in cancer and autoimmunity [6,8].
How EDITGENE Supports tRNA (cytidine-N5)-methyltransferase activity Research
Researchers studying tRNA (cytidine-N5)-methyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in a specific phenotype, which requires precise genetic models. EDITGENE provides a comprehensive suite of CRISPR services to generate such models efficiently and reproducibly.
Contact EDITGENE today to design your custom CRISPR model for tRNA (cytidine-N5)-methyltransferase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| NSUN2 Knockout HEK293 Cell Line | EDJ-KQ174 | Human | 54888 | Details Get a Quote |
| TRDMT1 Knockout HEK293 Cell Line | EDJ-KQ4474 | Human | 1787 | Details Get a Quote |
| NSUN6 Knockout HEK293 Cell Line | EDJ-KQ8795 | Human | 221078 | Details Get a Quote |
| NSUN3 Knockout HEK293 Cell Line | EDJ-KQ14502 | Human | 63899 | Details Get a Quote |
| NSUN2 Knockout A-549 Cell Line | EDJ-KQ19956 | Human | 54888 | Details Get a Quote |
| NSUN2 Knockout HCT 116 Cell Line | EDJ-KQ19957 | Human | 54888 | Details Get a Quote |
| NSUN2 Knockout HeLa Cell Line | EDJ-KQ19958 | Human | 54888 | Details Get a Quote |
| TRDMT1 Knockout A-549 Cell Line | EDJ-KQ27035 | Human | 1787 | Details Get a Quote |
| TRDMT1 Knockout HCT 116 Cell Line | EDJ-KQ27036 | Human | 1787 | Details Get a Quote |
| TRDMT1 Knockout HeLa Cell Line | EDJ-KQ27037 | Human | 1787 | Details Get a Quote |
| NSUN6 Knockout A-549 Cell Line | EDJ-KQ35082 | Human | 221078 | Details Get a Quote |
| NSUN6 Knockout HeLa Cell Line | EDJ-KQ35084 | Human | 221078 | Details Get a Quote |
| NSUN3 Knockout A-549 Cell Line | EDJ-KQ44769 | Human | 63899 | Details Get a Quote |
| NSUN3 Knockout HCT 116 Cell Line | EDJ-KQ44770 | Human | 63899 | Details Get a Quote |
| NSUN3 Knockout HeLa Cell Line | EDJ-KQ44771 | Human | 63899 | Details Get a Quote |
Displaying Records 1 To 15 Of 19 Records
Frequently Asked Questions About tRNA (cytidine-N5)-methyltransferase activity
What is tRNA (cytidine-N5)-methyltransferase activity?
It is the enzymatic activity that adds a methyl group to the N5 position of cytidine in tRNA, using S-adenosyl-L-methionine as the methyl donor, as defined by GO:0016428.
What genes are involved in tRNA (cytidine-N5)-methyltransferase activity?
Genes encoding cytosine methyltransferases such as NSUN2 and NSUN6, as well as related tRNA methyltransferases like METTL1 and TRMT6/TRMT61A, are involved in tRNA methylation pathways [1,3,4,7].
Which diseases are linked to tRNA methylation?
tRNA methylation dysregulation is linked to cancer, autoimmunity, aging, and metabolic disorders [1,2,5,7,8].
How can I study tRNA (cytidine-N5)-methyltransferase activity?
You can use CRISPR knockout, point mutation, knock-in, and overexpression models combined with RNA-seq, Ribo-seq, and mass spectrometry [5,6,8].
What is the role of METTL1 in tRNA methylation?
METTL1 is the catalytic subunit of the m7G tRNA methyltransferase complex, and its dysregulation affects translation, cancer, and immunity [1,4,8].
Does tRNA cytidine methylation affect translation?
Yes, tRNA modifications including cytidine methylation influence tRNA stability, decoding, and translation efficiency [1,3,4].
What are the target sites of tRNA cytidine methylation?
The modification can occur at cytidine(34), cytidine(40), cytidine(48), and cytidine(49) in tRNA.
Can CRISPR be used to study tRNA methyltransferases?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression are widely used to dissect tRNA methyltransferase function [5,6,8].
What is the difference between m5C and m7G in tRNA?
m5C is cytosine methylation at the N5 position, while m7G is methylation of guanosine at the N7 position; both are catalyzed by distinct enzymes and have different functions [1,3,4].
How does tRNA methylation contribute to cancer?
tRNA methylation can support metabolic reprogramming and oncogenic transformation, as shown for m7G and m1A pathways [2,6,7].
Conclusion
GO:0016428, tRNA (cytidine-N5)-methyltransferase activity, represents a fundamental RNA modification mechanism with broad implications for translation, cellular stress responses, and human disease. Continued research using precise CRISPR models and advanced sequencing technologies will further elucidate how this activity contributes to cancer, autoimmunity, and aging [1,2,5,7,8].
References
- 1. Li J et al.. 2023. Structural basis of regulated m(7)G tRNA modification by METTL1-WDR4.. Nature 613(7943):391-397 PMID: 36599985
- 2. Delaunay S et al.. 2022. Mitochondrial RNA modifications shape metabolic plasticity in metastasis.. Nature 607(7919):593-603 PMID: 35768510
- 3. Lyko F. 2018. The DNA methyltransferase family: a versatile toolkit for epigenetic regulation.. Nat Rev Genet 19(2):81-92 PMID: 29033456
- 4. Ruiz-Arroyo VM et al.. 2023. Structures and mechanisms of tRNA methylation by METTL1-WDR4.. Nature 613(7943):383-390 PMID: 36599982
- 5. Fu Y et al.. 2024. Perturbation of METTL1-mediated tRNA N(7)- methylguanosine modification induces senescence and aging.. Nat Commun 15(1):5713 PMID: 38977661
- 6. Ali RH et al.. 2025. A methyltransferase-independent role for METTL1 in tRNA aminoacylation and oncogenic transformation.. Mol Cell 85(5):948-961.e11 PMID: 39892392
- 7. Wang Y et al.. 2021. N(1)-methyladenosine methylation in tRNA drives liver tumourigenesis by regulating cholesterol metabolism.. Nat Commun 12(1):6314 PMID: 34728628
- 8. Wang S et al.. 2024. Aberrant METTL1-mediated tRNA m(7)G modification alters B-cell responses in systemic autoimmunity in humans and mice.. Nat Commun 15(1):10599 PMID: 39638793
