GO:0009982 pseudouridine synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0009982 pseudouridine synthase activity describes the enzymatic conversion of uridine to pseudouridine within RNA by rotating the glycosidic bond, a widespread post-transcriptional modification.
• Pseudouridine synthases (PUS enzymes) modify diverse RNA species including tRNA, mRNA, and tRNA-derived fragments, thereby influencing translation, RNA stability, and cellular stress responses.
• Dysregulation of pseudouridine synthase activity is implicated in cancers such as hepatocellular carcinoma and glioblastoma, where PUS1 and PUS7 promote oncogenic translation and tumor growth.
• PUS enzymes also play roles in antiviral immunity, hypoxia response, and retrotransposon-driven inflammation, highlighting their broad physiological impact.
• CRISPR-based knockout, point mutation, knock-in, and overexpression models are essential to dissect the causal roles of individual PUS genes in disease and RNA biology.
• Targeting pseudouridine synthase activity, for example with PUS7 inhibitors, represents a promising therapeutic strategy in oncology and other diseases.
Description
Pseudouridine synthase activity (GO:0009982) is a molecular function that catalyzes the isomerization of uridine to pseudouridine in RNA molecules. This modification, known as pseudouridylation, is one of the most abundant post-transcriptional RNA modifications and occurs across all domains of life. The enzyme rotates the C1'-N-1 glycosidic bond of uridine to a C1'-C5 bond, creating a carbon-carbon linkage that distinguishes pseudouridine from uridine. Researchers study this activity because it affects RNA structure, stability, and function, and its dysregulation is linked to cancer, stem cell biology, and immune responses. Understanding pseudouridine synthase activity at the molecular level provides insights into how cells fine-tune gene expression and respond to stress.
pseudouridine synthase activity At A Glance
| GO ID | GO:0009982 |
|---|---|
| GO term | pseudouridine synthase activity |
| Ontology | molecular_function |
| Synonym | None listed |
| Definition | Catalysis of the reaction: a uridine in RNA = a pseudouridine in RNA. Conversion of uridine in an RNA molecule to pseudouridine by rotation of the C1'-N-1 glycosidic bond of uridine in RNA to a C1'-C5. |
| Major function | Post-transcriptional RNA modification by isomerization of uridine to pseudouridine |
| EC number | 5.4.99.- (isomerase class) |
| Substrates | Uridine within tRNA, mRNA, rRNA, snRNA, and tRNA-derived fragments |
| Products | Pseudouridine within RNA and free pseudouridine after degradation |
What Is GO:0009982?
Pseudouridine synthase activity (GO:0009982) is defined as the catalysis of the reaction: a uridine in RNA = a pseudouridine in RNA. This conversion involves the rotation of the C1'-N-1 glycosidic bond of uridine in RNA to a C1'-C5 bond, resulting in the isomer pseudouridine. The reaction does not require ATP or other energy sources and is carried out by a family of enzymes known as pseudouridine synthases (PUS proteins).
Why Is pseudouridine synthase activity Important in Cell Biology?
Pseudouridine synthase activity is critical because pseudouridylation alters RNA structure and function, impacting translation, splicing, and RNA stability. This modification is essential for normal development and cellular homeostasis, and its dysregulation contributes to diseases including cancer, neurological disorders, and immune dysfunction. Studying this activity helps researchers understand fundamental RNA biology and develop therapeutic strategies targeting RNA-modifying enzymes.
• Pseudouridylation by PUS enzymes enhances translational fidelity and efficiency in stem cells and cancer cells.
• PUS7-mediated pseudouridylation of tRNA-derived fragments regulates protein synthesis and stem cell maintenance.
• PUS1 promotes hepatocellular carcinoma by modifying mRNA to enhance translation of oncogenic transcripts.
• PUS7 inhibition suppresses glioblastoma tumorigenesis, suggesting a therapeutic window.
• Pseudouridine synthase activity is involved in antiviral immunity and can boost cancer immunotherapy responses.
• Hypoxia-responsive tRNA-derived small RNAs require pseudouridylation for renal protection via RNA autophagy.
• PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation, linking pseudouridylation to innate immunity.
• Dysregulated pseudouridine synthases are emerging as biomarkers and drug targets in multiple cancers.
• Pseudouridine modification is crucial for the function of spliceosomal snRNAs and ribosome biogenesis.
• Understanding PUS enzyme specificity informs the design of RNA-based therapeutics and vaccines.
Molecular Mechanism of pseudouridine synthase activity
Substrate Recognition and Binding
In simple terms: The enzyme first grabs the RNA molecule and finds the specific uridine it needs to modify.
Pseudouridine synthases recognize their target uridines within RNA substrates through a combination of sequence and structural elements. For example, human PUS3 selectively modifies tRNA at position 39, relying on specific features of the tRNA elbow region. PUS1 can modify both tRNA and mRNA, and its activity on mRNA is linked to translation enhancement. The binding step ensures that only the correct uridine is targeted, preventing random modification.
Catalytic Isomerization
In simple terms: The enzyme then flips the chemical bond of uridine to turn it into pseudouridine.
The catalytic mechanism involves the rotation of the C1'-N-1 glycosidic bond of uridine to a C1'-C5 bond, forming pseudouridine. This isomerization is a multi-step process that typically requires a conserved aspartate residue in the active site. The reaction does not require ATP; instead, it relies on the enzyme's ability to stabilize the transition state. The product, pseudouridine, has an extra hydrogen bond donor that can stabilize RNA structure.
Release and RNA Folding
In simple terms: After modification, the RNA is released and can fold into its functional shape.
Once pseudouridine is formed, the modified RNA is released from the enzyme. The presence of pseudouridine can alter the local RNA structure, often increasing thermodynamic stability and affecting interactions with proteins or other RNAs. For instance, pseudouridylation of tRNA-derived fragments influences their function in translational control. This step is crucial for the downstream biological effects of pseudouridylation.
Enzyme Specificity and Regulation
In simple terms: Different enzymes modify different RNAs, and their activity can be turned up or down.
The PUS family comprises multiple enzymes with distinct substrate specificities. PUS1, PUS3, PUS7, and PUS10 each target different RNA types or positions. Their activity can be regulated at the expression level or through post-translational modifications, although specific regulatory mechanisms are still being elucidated. For example, PUS7 is overexpressed in glioblastoma and its inhibition reduces tumor growth. Hypoxia can induce specific tRNA-derived small RNAs that depend on pseudouridylation for their protective function.
Key Genes Involved in GO:0009982 pseudouridine synthase activity
The following genes encode pseudouridine synthases or are closely associated with pseudouridine synthase activity, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PUS1 | Pseudouridine synthase that modifies tRNA and mRNA | Promotes hepatocellular carcinoma via mRNA pseudouridylation; target for antiviral immunity |
| PUS7 | Pseudouridine synthase that modifies tRNA and tRNA-derived fragments | Suppresses glioblastoma tumorigenesis; regulates stem cell translational control |
| PUS3 | tRNA pseudouridine synthase specific for position 39 | Molecular basis of tRNA selectivity studied; linked to neurological disorders |
| PUS10 | Pseudouridine synthase involved in tRNA fragmentation | Impacts retrotransposon-driven inflammation |
| DKC1 | Component of telomerase and H/ACA ribonucleoprotein complex | Dyskeratosis congenita; pseudouridylation of rRNA and snRNA |
| NHP2 | H/ACA ribonucleoprotein complex component | Assists in pseudouridylation of rRNA; ribosomopathies |
| NOP10 | H/ACA ribonucleoprotein complex component | Required for pseudouridine synthase activity in ribosomes |
| GAR1 | H/ACA ribonucleoprotein complex component | Facilitates pseudouridylation of rRNA and snRNA |
| TRUB1 | Pseudouridine synthase for mRNA | Modifies mRNA and affects stability |
| PUS4 | tRNA pseudouridine synthase | Yeast homolog; model for studying pseudouridylation |
| PUS6 | Mitochondrial pseudouridine synthase | Modifies mitochondrial tRNAs |
| PUS9 | Mitochondrial pseudouridine synthase | Modifies mitochondrial tRNAs |
| RPUSD1 | RNA pseudouridine synthase domain containing 1 | Potential role in RNA modification |
| RPUSD2 | RNA pseudouridine synthase domain containing 2 | Potential role in RNA modification |
| RPUSD3 | RNA pseudouridine synthase domain containing 3 | Mitochondrial RNA modification |
| RPUSD4 | RNA pseudouridine synthase domain containing 4 | Mitochondrial RNA modification |
How Is pseudouridine synthase activity Regulated?
Pseudouridine synthase activity is regulated at multiple levels. Expression of PUS enzymes can be induced by cellular stress such as hypoxia, which leads to the production of specific tRNA-derived small RNAs that require pseudouridylation for their protective function. In cancer, PUS1 and PUS7 are often overexpressed, promoting oncogenic translation and tumor growth. Conversely, inhibition of PUS7 suppresses glioblastoma tumorigenesis, indicating that its activity is tightly controlled and can be targeted therapeutically. Additionally, PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation, suggesting a role in innate immune regulation. The H/ACA ribonucleoprotein complex, which includes DKC1, NHP2, NOP10, and GAR1, regulates pseudouridylation of rRNA and snRNA, and its dysfunction leads to diseases like dyskeratosis congenita.
pseudouridine synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PUS1 | Hepatocellular carcinoma; antiviral immunity | Knockout or overexpression in liver cancer cell lines; xenograft models |
| PUS7 | Glioblastoma; stem cell translational control | Knockout or inhibitor treatment in glioblastoma cell lines and patient-derived xenografts |
| PUS3 | Intellectual disability; tRNA modification | Point mutation knock-in in cell lines or animal models |
| PUS10 | Retrotransposon-driven inflammation | Knockout in immune cells; inflammation models |
| DKC1 | Dyskeratosis congenita; ribosomopathy | Knockout or point mutation in hematopoietic stem cells |
Cancer
Pseudouridine synthase activity is frequently dysregulated in cancer. PUS1 promotes hepatocellular carcinoma by pseudouridylating mRNA to enhance the translation of oncogenic mRNAs. PUS7 is overexpressed in glioblastoma and its inhibition suppresses tumorigenesis, making it a potential therapeutic target. Targeting PUS1 can also activate antiviral immunity and boost cancer immunotherapy. These findings highlight the oncogenic roles of pseudouridine synthases and the potential of inhibitors.
Neurological Disorders and Stem Cell Biology
Pseudouridylation of tRNA-derived fragments by PUS7 steers translational control in stem cells, affecting their differentiation and maintenance. Dysregulation of this process may contribute to neurological disorders, although direct links require further study. PUS3 mutations have been associated with intellectual disability, underscoring the importance of pseudouridine synthase activity in brain development.
Inflammation and Immunity
PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation, linking pseudouridine synthase activity to innate immune responses. Additionally, PUS1-targeted therapy activates antiviral immunity, suggesting a role in antiviral defense. Hypoxia-responsive tRNA-derived small RNAs confer renal protection through RNA autophagy, a process dependent on pseudouridylation.
From pseudouridine synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of PUS1 affect oncogenic translation in hepatocellular carcinoma? | PUS1 knockout in HepG2 or Huh7 cells; polysome profiling |
| Can PUS7 inhibition suppress glioblastoma growth? | PUS7 knockout or small molecule inhibitor in U87 or patient-derived glioblastoma cells |
| What is the role of PUS3 in tRNA modification and neuronal function? | PUS3 point mutation knock-in in iPSC-derived neurons |
| How does PUS10-mediated tRNA fragmentation impact inflammation? | PUS10 knockout in macrophages; cytokine profiling |
| Does pseudouridylation of tRNA-derived fragments regulate stem cell fate? | PUS7 knockout in embryonic stem cells; ribosome profiling |
| Can PUS1 overexpression boost antitumor immunity? | PUS1 overexpression in cancer cells; co-culture with T cells |
How to Study the pseudouridine synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Ribo-seq | Translation efficiency and ribosome occupancy | Assessing impact of PUS1 on oncogenic mRNA translation |
| Psi-seq | Global pseudouridylation sites | Mapping PUS7 targets in glioblastoma |
| Mass spectrometry | Pseudouridine quantification and protein interactions | Studying H/ACA complex function |
| CRISPR knockout screens | Gene essentiality and drug sensitivity | Identifying modifiers of PUS7 inhibitor response |
| Northern blot | tRNA fragmentation and modification | Analyzing PUS10-induced tRNA fragments |
| Polysome profiling | mRNA translation status | Evaluating PUS1 effects on oncogene translation |
| Immunoprecipitation | Protein-RNA interactions | Detecting PUS enzyme-substrate complexes |
| Luciferase reporter assays | Translational control | Measuring pseudouridine-dependent translation |
Ribosome Profiling (Ribo-seq)
Ribo-seq measures translation efficiency at codon resolution. It can reveal how pseudouridylation by PUS enzymes affects mRNA translation, as shown for PUS1 in hepatocellular carcinoma. This method is essential for understanding the functional consequences of pseudouridine synthase activity on protein synthesis.
RNA Sequencing and Modification Mapping
RNA-seq combined with pseudouridine-specific chemical labeling (e.g., Psi-seq) allows global mapping of pseudouridylation sites. Such approaches have identified PUS7 targets in glioblastoma and PUS1 targets in liver cancer. These techniques are crucial for determining the substrate specificity of PUS enzymes.
Mass Spectrometry and Proteomics
Mass spectrometry can quantify pseudouridine levels in RNA and identify proteins interacting with PUS enzymes. This method has been used to study the H/ACA ribonucleoprotein complex components required for pseudouridylation. Proteomics can also reveal downstream effects on protein expression.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes that modulate sensitivity to PUS inhibitors or regulate pseudouridylation. Such screens have been instrumental in uncovering the role of PUS7 in glioblastoma. They enable unbiased discovery of pathways linked to pseudouridine synthase activity.
How CRISPR Can Be Used to Study GO:0009982 pseudouridine synthase activity
Knockout
CRISPR knockout of PUS genes is used to abolish pseudouridine synthase activity and study loss-of-function phenotypes. For example, PUS7 knockout suppresses glioblastoma tumorigenesis, and PUS1 knockout reduces oncogenic translation in hepatocellular carcinoma. Knockout models are essential for validating the causal role of specific PUS enzymes.
Point Mutation
Point mutations in the catalytic domain of PUS enzymes can separate enzymatic activity from other functions. For instance, mutating the conserved aspartate in PUS3 abolishes pseudouridylation, allowing researchers to study the consequences of loss of activity without affecting protein stability. Such models are valuable for dissecting structure-function relationships.
Knock-in
Knock-in of tagged PUS genes (e.g., FLAG or GFP) enables localization and interaction studies. Tagged PUS3 has been used to study tRNA selectivity. Knock-in of disease-associated mutations, such as those in DKC1, can model dyskeratosis congenita.
Overexpression
Overexpression of PUS enzymes is used to investigate gain-of-function effects. PUS1 overexpression enhances translation of oncogenic mRNAs and promotes hepatocellular carcinoma. Overexpression models help identify downstream targets and potential therapeutic vulnerabilities.
How EDITGENE Supports pseudouridine synthase activity Research
Researchers studying pseudouridine synthase activity-related genes often need to determine whether a candidate gene is causally involved in RNA modification, translation control, or disease progression. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling rigorous functional studies.
Contact EDITGENE today to design your custom CRISPR model for pseudouridine synthase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| PUS1 Knockout HEK293 Cell Line | EDJ-KQ1951 | Human | 80324 | Details Get a Quote |
| PUS7 Knockout HEK293 Cell Line | EDJ-KQ2333 | Human | 54517 | Details Get a Quote |
| TRUB1 Knockout HEK293 Cell Line | EDJ-KQ3836 | Human | 142940 | Details Get a Quote |
| RPUSD1 Knockout HEK293 Cell Line | EDJ-KQ7406 | Human | 113000 | Details Get a Quote |
| RPUSD2 Knockout HEK293 Cell Line | EDJ-KQ8672 | Human | 27079 | Details Get a Quote |
| PUSL1 Knockout HEK293 Cell Line | EDJ-KQ8966 | Human | 126789 | Details Get a Quote |
| PUS7L Knockout HEK293 Cell Line | EDJ-KQ9843 | Human | 83448 | Details Get a Quote |
| PUS3 Knockout HEK293 Cell Line | EDJ-KQ9853 | Human | 83480 | Details Get a Quote |
| PUS10 Knockout HEK293 Cell Line | EDJ-KQ11316 | Human | 150962 | Details Get a Quote |
| RPUSD3 Knockout HEK293 Cell Line | EDJ-KQ15128 | Human | 285367 | Details Get a Quote |
| TRUB1 Knockout HCT 116 Cell Line | EDJ-KQ25991 | Human | 142940 | Details Get a Quote |
| RPUSD1 Knockout A-549 Cell Line | EDJ-KQ31204 | Human | 113000 | Details Get a Quote |
| RPUSD3 Knockout HCT 116 Cell Line | EDJ-KQ44486 | Human | 285367 | Details Get a Quote |
| PUS1 Knockout HeLa Cell Line | EDJ-KQ18329 | Human | 80324 | Details Get a Quote |
| TRUB1 Knockout HeLa Cell Line | EDC90504 | Human | 142940 | Details Get a Quote |
Displaying Records 1 To 15 Of 42 Records
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Frequently Asked Questions About pseudouridine synthase activity
What is pseudouridine synthase activity?
Pseudouridine synthase activity (GO:0009982) is the enzymatic conversion of uridine to pseudouridine in RNA by rotating the glycosidic bond, a key post-transcriptional modification.
What genes are involved in pseudouridine synthase activity?
Key genes include PUS1, PUS3, PUS7, PUS10, DKC1, NHP2, NOP10, GAR1, and RPUSD family members, each with distinct RNA targets.
How does pseudouridine synthase activity affect cancer?
Dysregulated PUS enzymes promote cancer; PUS1 enhances oncogenic translation in liver cancer, and PUS7 supports glioblastoma growth.
What diseases are linked to pseudouridine synthase mutations?
Mutations in PUS3 and DKC1 are associated with intellectual disability and dyskeratosis congenita, respectively.
Can pseudouridine synthase activity be targeted therapeutically?
Yes, PUS7 inhibitors suppress glioblastoma, and targeting PUS1 boosts cancer immunotherapy, showing therapeutic potential.
How is pseudouridine synthase activity measured?
Methods include Psi-seq for global mapping, mass spectrometry for quantification, and Ribo-seq for translation effects.
What is the role of pseudouridine in tRNA?
Pseudouridine stabilizes tRNA structure and function; PUS3 modifies tRNA at position 39, affecting translation.
Which pseudouridine synthase modifies mRNA?
PUS1 and TRUB1 can modify mRNA, influencing translation and stability.
How does hypoxia affect pseudouridine synthase activity?
Hypoxia induces tRNA-derived small RNAs that require pseudouridylation for renal protection via RNA autophagy.
What CRISPR models are available for studying pseudouridine synthases?
Knockout, point mutation, knock-in, and overexpression models can be custom-generated for any PUS gene.
Conclusion
Pseudouridine synthase activity (GO:0009982) is a fundamental RNA modification process with far-reaching implications in cancer, stem cell biology, and immunity. The growing understanding of PUS enzymes and their substrates offers new opportunities for therapeutic intervention. Continued research using advanced CRISPR models and RNA profiling technologies will further illuminate the roles of pseudouridylation in health and disease.
References
- 1. Hu YX et al.. 2024. Pseudouridine synthase 1 promotes hepatocellular carcinoma through mRNA pseudouridylation to enhance the translation of oncogenic mRNAs.. Hepatology 80(5):1058-1073 PMID: 38015993
- 2. Cui Q et al.. 2021. Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis.. Nat Cancer 2(9):932-949 PMID: 35121864
- 3. Li G et al.. 2025. A hypoxia-responsive tRNA-derived small RNA confers renal protection through RNA autophagy.. Science 389(6763):eadp5384 PMID: 40674449
- 4. Wang F et al.. 2025. Pseudouridine synthase 1-targeted therapy activates antiviral immunity to boost cancer immunotherapy.. Cell Rep 44(9):116233 PMID: 40911416
- 5. Guzzi N et al.. 2018. Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.. Cell 173(5):1204-1216.e26 PMID: 29628141
- 6. 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
- 7. Yang Q et al.. 2025. Pseudouridine Synthase 7 in Cancer: Functions, Mechanisms, and Therapeutic Potential.. Cells 14(17) PMID: 40940790
- 8. Madej M et al.. 2025. PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation.. Cell Rep 44(6):115735 PMID: 40402745
