GO:0102262 tRNA-dihydrouridine16 synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0102262 describes the molecular function of catalyzing the NAD(P)-dependent conversion of uracil16 in tRNA to 5,6-dihydrouracil16, producing NAD(P)H and a proton.
• This activity is a tRNA modification enzyme function that introduces dihydrouridine at position 16, a residue critical for tRNA structure and function.
• Dihydrouridine formation at position 16 is conserved and contributes to the thermodynamic stability and flexibility of the tRNA elbow region.
• Dysregulation of tRNA modifications, including dihydrouridine synthesis, has been linked to human diseases such as cancer and neurological disorders.
• Research on this activity employs CRISPR knockout, point mutation, and overexpression models to dissect gene function and substrate specificity.
• EDITGENE provides comprehensive CRISPR services, including knockout, knock-in, point mutation, overexpression, library screening, and bioinformatics, to accelerate tRNA modification research.
Description
GO:0102262, tRNA-dihydrouridine16 synthase activity, is a molecular function term that describes the enzymatic conversion of uracil at position 16 of tRNA into 5,6-dihydrouracil, using NAD(P) as a cofactor. This modification is part of the broader family of tRNA dihydrouridine synthases, which introduce dihydrouridine (D) residues into tRNA molecules, influencing their structure and function. The specific position 16 modification is crucial for the proper folding of the tRNA elbow region, affecting translation efficiency and fidelity. Researchers study this activity to understand its role in cellular physiology and its implications in diseases such as cancer and neurodegeneration. The reaction catalyzed by this enzyme is: a 5,6-dihydrouracil16 in tRNA + NAD(P) = H+ + a uracil16 in tRNA + NAD(P)H, as defined by QuickGO. This activity is essential for maintaining the dynamic landscape of tRNA modifications, which are increasingly recognized as key regulators of gene expression. Understanding GO:0102262 provides insights into the molecular mechanisms of translation regulation and offers potential therapeutic targets for diseases linked to tRNA modification defects.
tRNA-dihydrouridine16 synthase activity At A Glance
| GO ID | GO:0102262 |
|---|---|
| GO term | tRNA-dihydrouridine16 synthase activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Catalyzes the NAD(P)-dependent reduction of uracil16 in tRNA to 5,6-dihydrouracil16 |
| Reaction | a 5,6-dihydrouracil16 in tRNA + NAD(P) = H+ + a uracil16 in tRNA + NAD(P)H |
| Cofactor | NAD(P) |
| Substrate | tRNA containing uracil at position 16 |
| Product | tRNA containing 5,6-dihydrouracil at position 16 |
What Is GO:0102262?
tRNA-dihydrouridine16 synthase activity (GO:0102262) is defined as the catalysis of the reaction: a 5,6-dihydrouracil16 in tRNA + NAD(P) = H+ + a uracil16 in tRNA + NAD(P)H. In other words, this enzyme activity removes a uracil base at position 16 of a tRNA molecule and reduces it to 5,6-dihydrouracil, using NAD(P) as an electron donor and releasing NAD(P)H and a proton. This modification is a post-transcriptional change that alters the chemical nature of the nucleobase, impacting tRNA structure and interactions.
Why Is tRNA-dihydrouridine16 synthase activity Important in Cell Biology?
GO:0102262 is important because dihydrouridine modification at tRNA position 16 is a conserved post-transcriptional modification that affects tRNA stability, folding, and decoding capacity, thereby influencing global protein synthesis. Aberrations in tRNA modification enzymes have been associated with a range of human diseases, including cancer, mitochondrial disorders, and neurological diseases. Studying this activity helps elucidate the molecular basis of translation regulation and may reveal therapeutic vulnerabilities in diseases characterized by dysregulated tRNA modifications.
• Dihydrouridine at position 16 enhances tRNA structural flexibility, which is critical for efficient ribosomal translation.
• This modification is conserved across all domains of life, underscoring its fundamental role in tRNA biology.
• Altered expression of tRNA-dihydrouridine synthases has been observed in various cancers, suggesting a role in tumorigenesis.
• Mutations in tRNA modification enzymes can lead to neurological disorders and mitochondrial diseases.
• The activity requires NAD(P), linking tRNA modification to cellular redox and metabolic states.
• Understanding GO:0102262 can inform the design of inhibitors or modulators for therapeutic intervention.
• CRISPR-based models enable precise dissection of the physiological roles of this activity in vivo.
• Bioinformatics and library screening approaches can identify novel regulators and substrates of this enzyme.
What Happens During tRNA-dihydrouridine16 synthase activity?
Substrate Recognition and Binding
In simple terms: The enzyme finds and grabs the tRNA molecule at the right spot.
The enzyme specifically recognizes tRNA molecules and binds to the region containing uracil at position 16. This binding is mediated by structural elements within the tRNA, such as the D-arm, and ensures that the modification occurs at the correct location. The specificity for position 16 is determined by the enzyme's active site architecture and its interaction with the tRNA scaffold.
Catalytic Reduction of Uracil to Dihydrouracil
In simple terms: The enzyme uses NAD(P) to add hydrogen atoms to uracil, turning it into dihydrouracil.
Once bound, the enzyme catalyzes the reduction of the uracil base at position 16. This reaction involves the transfer of hydride from NAD(P)H to the uracil ring, forming 5,6-dihydrouracil. The reaction is: a 5,6-dihydrouracil16 in tRNA + NAD(P) = H+ + a uracil16 in tRNA + NAD(P)H. The enzyme likely employs a conserved catalytic mechanism involving a flavin or other cofactor, although the exact residues involved may vary among homologs.
Product Release and tRNA Recycling
In simple terms: After modification, the tRNA is released to perform its role in translation.
Following the modification, the dihydrouridine-containing tRNA is released from the enzyme. The modified tRNA can then participate in translation, where the dihydrouridine at position 16 contributes to the proper folding and stability of the tRNA. The enzyme is then free to catalyze another round of modification on a new substrate.
Key Genes Involved in GO:0102262 tRNA-dihydrouridine16 synthase activity
The following genes encode enzymes or associated factors that carry out or regulate tRNA-dihydrouridine16 synthase activity, as supported by published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DUS1 | tRNA-dihydrouridine synthase 1 | Catalyzes dihydrouridine formation at position 16 in tRNA; studied in yeast and humans |
| DUS2 | tRNA-dihydrouridine synthase 2 | Involved in dihydrouridine synthesis at multiple positions; linked to cancer |
| DUS3 | tRNA-dihydrouridine synthase 3 | Modifies tRNA at position 47; may have overlapping functions |
| DUS4 | tRNA-dihydrouridine synthase 4 | Catalyzes dihydrouridine at position 20; distinct from position 16 |
| tRNA | Substrate for modification | Specific tRNA species with uracil at position 16 are substrates |
| NAD(P) | Cofactor | Provides reducing equivalents for the reduction reaction |
| NAD(P)H | Product | Generated during the reaction; can influence cellular redox |
| H+ | Product | Proton released during the reaction |
| 5,6-dihydrouracil | Modified base | The product of the reaction; affects tRNA structure |
| Uracil | Substrate base | The target base at position 16 |
| Dihydrouridine synthase family | Enzyme family | Conserved proteins with diverse tRNA modification roles |
| tRNA modification machinery | Complex network | Includes other enzymes that modify tRNA; potential crosstalk |
| Translation apparatus | Downstream effector | Ribosomes and translation factors that utilize modified tRNA |
| Cellular redox regulators | Modulators | NAD(P)/NAD(P)H ratio affects enzyme activity |
| Disease-associated genes | Pathological links | Mutations in tRNA modification genes linked to diseases |
| CRISPR targets | Research tools | Genes edited to study function |
| Bioinformatics pipelines | Analysis tools | Identify homologs and predict substrates |
How Is tRNA-dihydrouridine16 synthase activity Regulated?
The activity of tRNA-dihydrouridine16 synthase is regulated at multiple levels. Expression of the encoding genes can be controlled transcriptionally in response to cellular stress or metabolic cues. The availability of cofactors NAD(P) and NAD(P)H influences enzymatic activity, linking tRNA modification to cellular redox status. Additionally, post-translational modifications of the enzyme may modulate its activity or localization. Substrate availability, i.e., the pool of tRNA molecules with uracil at position 16, also affects the overall modification rate.
tRNA-dihydrouridine16 synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DUS2 | Cancer (e.g., lung, breast) | CRISPR knockout in cancer cell lines; xenograft models |
| DUS1 | Neurological disorders | Knockout mice; neuronal cell cultures |
| DUS3 | Mitochondrial dysfunction | Patient-derived fibroblasts; mitochondrial assays |
| DUS4 | Developmental defects | Zebrafish knockouts; organoid models |
| tRNA modification genes | Ribosomopathies | Induced pluripotent stem cells; ribosome profiling |
Cancer
Dysregulation of tRNA modification enzymes, including dihydrouridine synthases, has been implicated in cancer. Altered expression of DUS genes can affect translation fidelity and promote tumorigenesis. For example, overexpression of DUS2 has been observed in certain cancers and is associated with poor prognosis. Targeting tRNA modification pathways may offer novel therapeutic strategies.
Neurological Disorders
Mutations in tRNA modification enzymes can cause neurological disorders such as intellectual disability and neurodegeneration. Defects in dihydrouridine synthesis may lead to impaired translation of key neuronal proteins, contributing to disease pathology. Understanding the role of GO:0102262 in neurons could reveal new targets for intervention.
Mitochondrial Diseases
Mitochondrial tRNA modifications are essential for mitochondrial translation. Defects in dihydrouridine formation at position 16 in mitochondrial tRNA could impair oxidative phosphorylation, leading to mitochondrial diseases. Research into this activity may shed light on the molecular mechanisms of these disorders.
From tRNA-dihydrouridine16 synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the catalytic mechanism of DUS1? | Point mutation of catalytic residues; in vitro enzyme assays |
| What are the physiological consequences of DUS2 loss? | CRISPR knockout in cell lines and mouse models |
| How does dihydrouridine16 affect tRNA structure? | Knock-in of modified tRNA; structural studies |
| Can DUS overexpression drive tumorigenesis? | Overexpression in cancer cell lines; xenografts |
| What are the interaction partners of DUS enzymes? | Tagged knock-in for affinity purification; proteomics |
| Which tRNAs are substrates for DUS4? | CRISPR library screening; RNA-seq |
How to Study the tRNA-dihydrouridine16 synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Ribo-seq | Translation efficiency and ribosome occupancy | Assess impact of DUS knockout on global translation |
| RNA-seq | Gene expression and tRNA levels | Identify changes in DUS gene expression in disease |
| Mass spectrometry | Dihydrouridine levels in tRNA | Validate enzyme activity and substrate specificity |
| CRISPR screening | Gene function and interactions | Discover regulators of tRNA modification |
| Western blot | Protein expression | Confirm knockout or overexpression of DUS genes |
| Immunoprecipitation | Protein-protein interactions | Identify DUS binding partners |
| In vitro enzyme assay | Catalytic activity | Measure kinetic parameters of DUS enzymes |
| Structural biology (cryo-EM, X-ray) | 3D structure of enzyme-tRNA complex | Understand catalytic mechanism |
Ribosome Profiling (Ribo-seq)
Ribo-seq measures translation efficiency and can reveal how dihydrouridine16 modification affects codon-specific translation. Knockout or overexpression of DUS genes followed by Ribo-seq can identify changes in ribosome occupancy.
RNA Sequencing (RNA-seq)
RNA-seq can detect changes in tRNA expression levels and modifications indirectly. Differential expression of DUS genes or tRNA species can be assessed in disease models.
Mass Spectrometry
Mass spectrometry can directly quantify dihydrouridine levels in tRNA. This method is used to validate the activity of DUS enzymes and to profile modifications in different conditions.
CRISPR Screening
Genome-wide CRISPR screens can identify genes that regulate or are regulated by tRNA-dihydrouridine16 synthase activity. This approach is powerful for discovering novel components of the tRNA modification network.
How CRISPR Can Be Used to Study GO:0102262 tRNA-dihydrouridine16 synthase activity
Knockout
CRISPR knockout of DUS genes allows researchers to study the loss-of-function phenotype. For example, knocking out DUS1 in cell lines can reveal its role in tRNA modification and translation. Knockout models are essential for validating gene function and identifying compensatory pathways.
Point Mutation
Introducing point mutations in the catalytic residues of DUS enzymes via CRISPR can dissect the enzymatic mechanism. For instance, mutating the NAD(P)-binding site can abolish activity and reveal substrate specificity. Point mutation models are valuable for structure-function studies.
Knock-in
Knock-in of tagged DUS genes (e.g., FLAG or GFP) enables visualization and purification of the enzyme. This approach helps track localization and interactions in live cells. Knock-in models can also be used to express mutant variants at endogenous levels.
Overexpression
CRISPR activation (CRISPRa) or cDNA overexpression can drive high levels of DUS enzymes to study gain-of-function effects. Overexpression models are useful for identifying downstream targets and potential oncogenic roles.
How EDITGENE Supports tRNA-dihydrouridine16 synthase activity Research
Researchers studying tRNA-dihydrouridine16 synthase activity-related genes often need to determine whether a candidate gene is causally involved in tRNA modification, translation regulation, or disease. EDITGENE provides a comprehensive suite of CRISPR services to enable precise genetic manipulation and functional interrogation of these genes.
Contact EDITGENE today to design your custom CRISPR model for tRNA-dihydrouridine16 synthase activity research.
Frequently Asked Questions About tRNA-dihydrouridine16 synthase activity
What is GO:0102262?
GO:0102262 is the Gene Ontology term for tRNA-dihydrouridine16 synthase activity, which catalyzes the NAD(P)-dependent reduction of uracil16 in tRNA to 5,6-dihydrouracil16.
What genes are involved in tRNA-dihydrouridine16 synthase activity?
Genes encoding dihydrouridine synthases such as DUS1, DUS2, DUS3, and DUS4 are involved in this activity, with DUS1 and DUS2 being the primary enzymes for position 16 modification.
What is the reaction catalyzed by tRNA-dihydrouridine16 synthase?
The reaction is: a 5,6-dihydrouracil16 in tRNA + NAD(P) = H+ + a uracil16 in tRNA + NAD(P)H.
Why is dihydrouridine at position 16 important?
Dihydrouridine at position 16 enhances tRNA structural flexibility and stability, which is critical for efficient translation and proper tRNA function.
How is tRNA-dihydrouridine16 synthase activity studied?
It is studied using methods such as CRISPR knockout, point mutation, overexpression, in vitro enzyme assays, mass spectrometry, and Ribo-seq.
What diseases are associated with defects in tRNA-dihydrouridine16 synthase activity?
Defects have been linked to cancer, neurological disorders, and mitochondrial diseases.
Can CRISPR be used to study tRNA-dihydrouridine16 synthase activity?
Yes, CRISPR knockout, knock-in, point mutation, and overexpression models are powerful tools to dissect the function of DUS genes.
What cofactors are required for tRNA-dihydrouridine16 synthase activity?
NAD(P) is required as a cofactor, providing reducing equivalents for the reduction of uracil to dihydrouracil.
Which tRNA position is modified by GO:0102262?
GO:0102262 specifically modifies position 16 of tRNA, converting uracil to 5,6-dihydrouracil.
How does EDITGENE support research on tRNA-dihydrouridine16 synthase activity?
EDITGENE provides CRISPR knockout, point mutation, knock-in, overexpression, library screening, and bioinformatics services to accelerate functional studies.
Conclusion
GO:0102262, tRNA-dihydrouridine16 synthase activity, represents a critical enzymatic function in tRNA modification that impacts translation and cellular physiology. Understanding its mechanism, regulation, and disease associations is essential for advancing molecular biology and developing therapeutic strategies. EDITGENE offers comprehensive CRISPR solutions to facilitate research on this important activity.
References
- 1. Sun S et al.. 2025. Exercise-induced histone lactylation in monocyte-derived macrophages restores cardiac immune homeostasis and function in sepsis-induced cardiomyopathy.. Nat Commun 17(1):756 PMID: 41398160
