GO:0160148 tRNA pseudouridine(55) synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0160148 describes the molecular function that catalyzes the isomerization of uridine(55) in tRNA to pseudouridine(55), a conserved modification critical for tRNA structure and function.
• The reaction is performed by pseudouridine synthases, with human PUS3 (and its homologs) acting as the primary tRNA pseudouridine(55) synthase.
• Pseudouridine(55) stabilizes the tRNA T-loop, influencing tRNA stability, translation fidelity, and cellular stress responses.
• Dysregulation of tRNA pseudouridine(55) synthase activity is linked to stem cell function, cancer progression, and antiviral immunity.
• Recent studies have mapped tRNA pseudouridylation landscapes, revealing that stand-alone pseudouridine synthases, including PUS3, have specific tRNA targets.
• CRISPR-based models (knockout, point mutation, knock-in, overexpression) are essential to dissect the causal roles of tRNA pseudouridine(55) synthase in disease and development.
Description
tRNA pseudouridine(55) synthase activity (GO:0160148) is a molecular function that catalyzes the conversion of uridine at position 55 of tRNA to pseudouridine (Ψ55). This modification is one of the most conserved post-transcriptional changes in tRNA and is critical for maintaining the canonical L-shaped tRNA structure and function. The enzyme responsible belongs to the pseudouridine synthase family, with human PUS3 being a well-characterized tRNA pseudouridine(55) synthase. Researchers study this activity because it impacts translation, stem cell biology, and disease mechanisms, including cancer and immune regulation.
tRNA pseudouridine(55) synthase activity At A Glance
| GO ID | GO:0160148 |
|---|---|
| GO term | tRNA pseudouridine(55) synthase activity |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Catalyzes the isomerization of uridine(55) to pseudouridine(55) in tRNA |
| Reaction | uridine(55) in tRNA = pseudouridine(55) in tRNA |
| Enzyme class | Pseudouridine synthase (isomerase) |
| Representative human gene | PUS3 (and possibly PUS1, PUS10 in related contexts) |
| Cellular role | tRNA modification, translation regulation, tRNA stability |
What Is GO:0160148?
GO:0160148 is defined as the catalysis of the reaction: uridine(55) in tRNA = pseudouridine(55) in tRNA. In other words, it is the enzymatic activity that isomerizes the uracil base of uridine at position 55 of a tRNA molecule into pseudouridine, a C-glycoside isomer.
Why Is tRNA pseudouridine(55) synthase activity Important in Cell Biology?
tRNA pseudouridine(55) synthase activity is essential for tRNA function and cellular homeostasis. The Ψ55 modification stabilizes the tRNA T-loop, which is crucial for tRNA folding and interaction with the ribosome. Disruption of this activity affects translation efficiency and fidelity, and has been implicated in stem cell maintenance, cancer progression, and immune responses. Understanding this activity provides insights into basic tRNA biology and potential therapeutic targets.
• Maintains tRNA structural integrity and stability through Ψ55 modification.
• Regulates translation and protein synthesis under normal and stress conditions.
• Influences stem cell self-renewal and differentiation via tRNA-derived fragments.
• Linked to cancer progression, including prostate cancer bone metastasis and immunotherapy responses.
• Modulates antiviral immunity and retrotransposon-driven inflammation.
• Provides a target for CRISPR-based functional studies and drug discovery.
• Conserved across species, making it a model for studying RNA modification enzymes.
• Dysregulation may contribute to ribosomopathies and neurological disorders.
Molecular Mechanism of tRNA pseudouridine(55) synthase activity
Substrate Recognition and Binding
In simple terms: The enzyme finds and grabs the tRNA molecule at the right spot.
tRNA pseudouridine(55) synthase specifically recognizes the T-arm of tRNA, binding to the elbow region where uridine 55 is located. Structural studies show that the enzyme makes direct contacts with the tRNA backbone and bases, ensuring selectivity for U55.
Catalytic Isomerization
In simple terms: The enzyme chemically changes uridine into pseudouridine.
The catalytic mechanism involves cleavage of the glycosidic bond between uracil and ribose, rotation of the base, and re-formation of a carbon-carbon bond, converting uridine to pseudouridine. This isomerization is characteristic of pseudouridine synthases and requires conserved aspartate residues in the active site.
Cofactors and Energy Requirements
In simple terms: No extra energy molecules are needed; the enzyme does the trick by itself.
Pseudouridine synthases, including tRNA pseudouridine(55) synthase, do not require ATP or other cofactors for catalysis; the reaction is an isomerization that proceeds without external energy input.
Regulation and Post-Translational Modifications
In simple terms: The enzyme's activity can be turned up or down by other cellular signals.
The activity of tRNA pseudouridine(55) synthase can be regulated at the expression level and potentially by post-translational modifications. For example, PUS1, a related pseudouridine synthase, is regulated by FOXA1 and affects EIF3b stability in a non-enzymatic pathway. Similar regulatory mechanisms may exist for PUS3, though direct evidence is limited.
Impact on tRNA Structure and Function
In simple terms: The modification makes tRNA more stable and helps it work better in protein synthesis.
Ψ55 stabilizes the T-loop through hydrogen bonding and stacking interactions, which is essential for the canonical L-shaped tRNA structure. This modification enhances tRNA thermostability and facilitates interactions with the ribosome during translation.
Key Genes Involved in GO:0160148 tRNA pseudouridine(55) synthase activity
The following genes and proteins are directly or indirectly involved in tRNA pseudouridine(55) synthase activity and its biological effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PUS3 | Primary tRNA pseudouridine(55) synthase in humans | Key enzyme for Ψ55 modification; knockout affects tRNA stability and translation |
| PUS1 | Pseudouridine synthase with broader tRNA targets; may compensate or interact | Linked to prostate cancer and antiviral immunity; non-enzymatic roles |
| PUS10 | Pseudouridine synthase involved in tRNA fragmentation | Impacts retrotransposon-driven inflammation |
| DKC1 | Pseudouridine synthase in H/ACA snoRNPs; not directly Ψ55 but related | Dyskeratosis congenita and ribosomopathies |
| TRUB1 | tRNA pseudouridine synthase for other positions | Comparative studies of tRNA modification |
| PUS7 | Pseudouridine synthase for tRNA and other RNAs | Stem cell function and translation control |
| EIF3b | Translation initiation factor | Stability regulated by PUS1 in cancer |
| FOXA1 | Transcription factor | Regulates PUS1 expression in prostate cancer |
| tRNA | Substrate for modification | Central to translation and cellular stress responses |
| Ribosome | Translation machinery | Interacts with modified tRNA during protein synthesis |
| PUS3 homologs (yeast) | Model for studying Ψ55 synthase | Conserved mechanism and substrate recognition |
| PUS4 (yeast) | Yeast Ψ55 synthase | Structural and biochemical studies |
| PUS1 (yeast) | Yeast pseudouridine synthase | Functional studies of tRNA modification |
| PUS7 (human) | Pseudouridine synthase for tRNA fragments | Stem cell translational control |
| PUS10 (human) | tRNA fragmentation and inflammation | Retrotransposon regulation |
| DKC1 (human) | H/ACA pseudouridine synthase | Ribosomopathy and cancer |
| NHP2 | H/ACA snoRNP component | Pseudouridylation complex assembly |
| GAR1 | H/ACA snoRNP component | Pseudouridylation complex assembly |
How Is tRNA pseudouridine(55) synthase activity Regulated?
The activity of tRNA pseudouridine(55) synthase is primarily regulated at the level of enzyme expression and potentially by post-translational modifications. For instance, PUS1 expression is controlled by FOXA1 in prostate cancer, and its stability affects EIF3b independently of its catalytic activity. Additionally, pseudouridine synthases can be regulated by cellular stress and immune signaling, as seen with PUS1-targeted therapy activating antiviral immunity. However, direct regulation of PUS3, the main Ψ55 synthase, remains less characterized.
tRNA pseudouridine(55) synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PUS1 | Prostate cancer bone metastasis; antiviral immunity | PUS1 knockout and overexpression in prostate cancer cell lines |
| PUS3 | tRNA modification defects; potential cancer and developmental disorders | PUS3 knockout in HEK293 or cancer cells |
| PUS7 | Stem cell differentiation and translation control | PUS7 knockout in embryonic stem cells |
| PUS10 | Retrotransposon-driven inflammation | PUS10 knockout in immune cells |
| DKC1 | Dyskeratosis congenita and ribosomopathy | DKC1 mutant knock-in models |
Cancer Progression and Metastasis
Dysregulation of tRNA pseudouridine(55) synthase activity has been implicated in cancer. PUS1, a related pseudouridine synthase, regulates EIF3b stability and promotes prostate cancer bone metastasis in a non-enzymatic pathway. Targeting PUS1 activates antiviral immunity and boosts cancer immunotherapy, suggesting that pseudouridine synthases are potential therapeutic targets.
Stem Cell Function and Differentiation
Pseudouridylation of tRNA-derived fragments by PUS7 steers translational control in stem cells, affecting self-renewal and differentiation. Although PUS3 specifically modifies tRNA at position 55, the broader family of pseudouridine synthases plays critical roles in stem cell biology.
Inflammation and Retrotransposon Control
PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation, linking pseudouridine synthase activity to innate immune responses. This suggests that tRNA modifications, including Ψ55, may influence inflammatory pathways.
Ribosomopathies and Neurodegeneration
Defects in pseudouridine synthases, such as DKC1, cause dyskeratosis congenita and other ribosomopathies. While direct links between PUS3 and neurodegeneration are not yet established, the importance of tRNA modifications in neuronal function suggests a potential role.
From tRNA pseudouridine(55) synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PUS3 knockout affect tRNA stability and translation? | PUS3 knockout cell lines (e.g., HEK293, HeLa) |
| What is the catalytic mechanism of U55 isomerization? | Point mutations in catalytic residues of PUS3 |
| How does Ψ55 modification influence tRNA structure? | Knock-in of tagged PUS3 for structural studies |
| Can PUS1 overexpression drive cancer metastasis? | PUS1 overexpression in prostate cancer cells |
| Does PUS10 loss affect retrotransposon silencing? | PUS10 knockout in macrophages |
| What is the role of Ψ55 in stem cell differentiation? | PUS3 knockout in induced pluripotent stem cells |
How to Study the tRNA pseudouridine(55) synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Ribo-seq | Translation efficiency and ribosome pausing | Global effects of PUS3 knockout on protein synthesis |
| Ψ-seq / Pseudo-seq | Pseudouridine sites in RNA | Mapping Ψ55 and other modifications |
| Mass spectrometry | Protein interactions and stability | Identifying PUS1-EIF3b interaction |
| X-ray crystallography | 3D structure of enzyme-tRNA complex | Understanding catalytic mechanism |
| CRISPR knockout | Loss-of-function phenotypes | Studying PUS3 role in tRNA modification |
| CRISPR knock-in | Tagged or mutant protein expression | Localizing PUS3 and studying dynamics |
| RNA immunoprecipitation (RIP) | RNA-protein interactions | Identifying tRNA targets of PUS3 |
| Polysome profiling | Ribosome-mRNA association | Assessing translation changes upon PUS3 loss |
Ribosome Profiling (Ribo-seq)
Ribo-seq measures translation efficiency and ribosome occupancy on mRNAs. It can reveal how loss of tRNA pseudouridine(55) synthase activity affects global translation and codon-specific pausing.
RNA Sequencing and Modification Mapping
RNA-seq and specialized techniques like Ψ-seq or Pseudo-seq can map pseudouridine sites in tRNA and other RNAs. These methods help identify the specific targets of PUS3 and other pseudouridine synthases.
Proteomics and Interactomics
Mass spectrometry-based proteomics can identify proteins interacting with PUS3 or affected by its loss. For example, PUS1 regulates EIF3b stability, which can be detected by Western blot or proteomics.
Structural Biology (X-ray Crystallography, Cryo-EM)
Structural studies of tRNA pseudouridine(55) synthase in complex with tRNA reveal substrate recognition and catalytic mechanisms. These methods provide atomic-level insights into the enzyme's function.
How CRISPR Can Be Used to Study GO:0160148 tRNA pseudouridine(55) synthase activity
Knockout
CRISPR knockout of PUS3 or related pseudouridine synthases allows researchers to study the loss of Ψ55 modification. This can reveal effects on tRNA stability, translation, and cellular phenotypes such as proliferation or stress response.
Point Mutation
Introducing point mutations in the catalytic residues of PUS3 (e.g., aspartate) via CRISPR can separate enzymatic activity from non-enzymatic functions. This is crucial for understanding the specific contribution of Ψ55 synthase activity.
Knock-in
Knock-in of tagged PUS3 (e.g., FLAG or GFP) enables localization, immunoprecipitation, and interaction studies. It also allows for the study of mutant versions of the enzyme in a physiological context.
Overexpression
Overexpression of PUS3 or PUS1 using CRISPR activation or lentiviral vectors can model gain-of-function effects, such as those observed in cancer. This helps identify downstream pathways and potential therapeutic targets.
How EDITGENE Supports tRNA pseudouridine(55) synthase activity Research
Researchers studying tRNA pseudouridine(55) synthase activity-related genes often need to determine whether a candidate gene is causally involved in tRNA modification, translation, or disease. EDITGENE provides comprehensive CRISPR services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for tRNA pseudouridine(55) synthase activity research.
Frequently Asked Questions About tRNA pseudouridine(55) synthase activity
What is tRNA pseudouridine(55) synthase activity?
It is the enzymatic activity that converts uridine at position 55 of tRNA to pseudouridine, as defined by GO:0160148.
What genes are involved in tRNA pseudouridine(55) synthase activity?
The primary human gene is PUS3, but related pseudouridine synthases like PUS1, PUS7, and PUS10 also play roles in tRNA modification.
What is the function of pseudouridine(55) in tRNA?
Ψ55 stabilizes the tRNA T-loop, which is essential for tRNA structure and translation.
How is tRNA pseudouridine(55) synthase activity regulated?
It is regulated at the expression level and potentially by post-translational modifications, as seen with PUS1 regulation by FOXA1.
What diseases are associated with tRNA pseudouridine(55) synthase activity?
Dysregulation is linked to cancer, stem cell defects, and inflammation, though direct links for PUS3 are still emerging.
How can I study tRNA pseudouridine(55) synthase activity?
Use CRISPR knockout, point mutation, knock-in, and overexpression models combined with Ribo-seq and modification mapping.
What is the reaction catalyzed by tRNA pseudouridine(55) synthase?
The isomerization of uridine(55) in tRNA to pseudouridine(55).
Is tRNA pseudouridine(55) synthase activity conserved?
Yes, it is highly conserved from yeast to humans, with PUS3 and its homologs performing the modification.
What are the research methods for studying Ψ55 modification?
Methods include Ribo-seq, Ψ-seq, mass spectrometry, and structural biology.
Can CRISPR be used to model tRNA pseudouridine(55) synthase dysfunction?
Yes, CRISPR knockout, point mutation, and knock-in models are powerful tools for studying this activity.
Conclusion
tRNA pseudouridine(55) synthase activity (GO:0160148) is a fundamental molecular function that ensures proper tRNA structure and translation. Its dysregulation is increasingly linked to cancer, stem cell biology, and immune responses. Continued research using CRISPR models and advanced sequencing techniques will further illuminate its roles and therapeutic potential.
References
- 1. Lin TY et al.. 2024. The molecular basis of tRNA selectivity by human pseudouridine synthase 3.. Mol Cell 84(13):2472-2489.e8 PMID: 38996458
- 2. Guzzi N et al.. 2018. Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.. Cell 173(5):1204-1216.e26 PMID: 29628141
- 3. Madej M et al.. 2025. PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation.. Cell Rep 44(6):115735 PMID: 40402745
- 4. Gu X et al.. 1998. Molecular recognition of tRNA by tRNA pseudouridine 55 synthase.. Biochemistry 37(1):339-43 PMID: 9425055
- 5. Wu Y et al.. 2024. FOXA1-dependent PUS1 regulates EIF3b stability in a non-enzymatic pathway mediating prostate cancer bone metastasis.. Int J Biol Sci 20(11):4566-4584 PMID: 39247811
- 6. Wang F et al.. 2025. Pseudouridine synthase 1-targeted therapy activates antiviral immunity to boost cancer immunotherapy.. Cell Rep 44(9):116233 PMID: 40911416
- 7. Hamma T et al.. 2006. Pseudouridine synthases.. Chem Biol 13(11):1125-35 PMID: 17113994
- 8. Xu H et al.. 2025. A comprehensive tRNA pseudouridine map uncovers targets dependent on human stand-alone pseudouridine synthases.. Nat Cell Biol 27(12):2186-2197 PMID: 41136621
