GO:0033897 ribonuclease T2 activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0033897 ribonuclease T2 activity is a molecular function describing a two-stage endonucleolytic cleavage of RNA to nucleoside 3'-phosphates and 3'-phosphooligonucleotides via 2',3'-cyclic phosphate intermediates.
• RNase T2 enzymes are non-base-specific endoribonucleases that function in acidic environments and are widely conserved from plants to humans.
• In mammals, lysosomal RNase T2 cooperates with PLD exonucleases to generate RNA ligands for TLR7 activation, linking this activity to innate immune sensing.
• RNase T2 restricts TLR13-mediated autoinflammation in vivo, and loss of function can lead to autoinflammatory pathology.
• Pseudouridine-modified RNA avoids immune detection through impaired endolysosomal processing and reduced TLR engagement, a process dependent on RNase T2 activity.
• In yeast, RNase T2 mediates selective autophagy of ribosomes induced by starvation, connecting this activity to ribosome quality control and nutrient stress responses.
Description
Ribonuclease T2 activity (GO:0033897) is a molecular function defined by the catalysis of a two-stage endonucleolytic cleavage of RNA, producing nucleoside 3'-phosphates and 3'-phosphooligonucleotides through 2',3'-cyclic phosphate intermediates. This activity is non-base-specific and is typically associated with acidic pH optima, distinguishing it from many other ribonucleases. The T2 family of ribonucleases is widely distributed across eukaryotes, bacteria, and viruses, and members play diverse roles in RNA turnover, host defense, and immune regulation. In recent years, the importance of RNase T2 in human health and disease has become increasingly clear. For example, lysosomal RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation, a critical step in innate immune sensing of RNA. Moreover, RNase T2 restricts TLR13-mediated autoinflammation in vivo, and its deficiency can lead to aberrant immune activation. In plants, T2 ribonucleases such as tomato LE are involved in pathogen responses, and secreted RNase T2 proteins from Fusarium species contribute to virulence. In yeast, RNase T2 mediates selective autophagy of ribosomes under starvation conditions. These findings underscore the broad biological significance of this enzyme activity and its relevance to immunology, microbiology, and cell biology. Researchers studying RNase T2 activity need reliable tools to manipulate and characterize the genes encoding these enzymes, making CRISPR-based models and functional genomics approaches essential.
ribonuclease T2 activity At A Glance
| GO ID | GO:0033897 |
|---|---|
| GO term | ribonuclease T2 activity |
| Ontology | molecular_function |
| Synonym | acid ribonuclease activity; acid RNase activity; base-non-specific ribonuclease activity; Escherichia coli ribonuclease II activity; ribonuclease II activity; RNase T2 activity; and others |
| Major function | Two-stage endonucleolytic cleavage of RNA to nucleoside 3'-phosphates and 3'-phosphooligonucleotides via 2',3'-cyclic phosphate intermediates |
| Reaction | a ribonucleotidyl-ribonucleotide-RNA + H2O = a 3'-end 3'-phospho-ribonucleotide-RNA + a 5'-end dephospho-ribonucleoside-RNA + H+ |
| Substrate specificity | Non-base-specific; cleaves RNA irrespective of nucleotide sequence |
| Cofactors | No known specific cofactors; activity is typically metal-independent |
| Localization | Often lysosomal or vacuolar in eukaryotes; secreted in some fungi and plants |
What Is GO:0033897?
GO:0033897 ribonuclease T2 activity describes the catalysis of a reaction in which a ribonucleotidyl-ribonucleotide-RNA is hydrolyzed in a two-stage endonucleolytic process. The first stage yields a 3'-end 3'-phospho-ribonucleotide-RNA and a 5'-end dephospho-ribonucleoside-RNA, with the formation of a 2',3'-cyclic phosphate intermediate. The second stage hydrolyzes this intermediate to produce a 3'-phospho-ribonucleotide and a 5'-dephospho-ribonucleoside. This activity is non-base-specific, meaning it cleaves RNA regardless of the nucleotide sequence, and it often functions optimally at acidic pH.
Why Is ribonuclease T2 activity Important in Cell Biology?
Ribonuclease T2 activity is critical for RNA degradation and turnover, innate immune sensing of RNA, and cellular stress responses. Its ability to generate RNA ligands for TLR7 and TLR13 places it at the center of autoimmune and autoinflammatory pathways. In plants and fungi, RNase T2 proteins contribute to pathogen virulence and host defense, affecting crop health and disease outcomes. In yeast, RNase T2 is required for selective autophagy of ribosomes during starvation, linking RNA degradation to nutrient sensing and ribosome quality control. Furthermore, pseudouridine-modified RNA evades immune detection by impairing endolysosomal processing and TLR engagement, a mechanism that depends on RNase T2 activity. Thus, understanding this enzyme is essential for immunology, microbiology, and RNA biology.
• RNase T2 generates RNA ligands for TLR7 activation in lysosomes, linking RNA degradation to innate immunity.
• RNase T2 restricts TLR13-mediated autoinflammation, and its loss causes autoinflammatory disease in vivo.
• Pseudouridine-modified RNA avoids immune detection through impaired endolysosomal processing and TLR engagement, a process involving RNase T2.
• In plants, tomato T2 ribonuclease LE is involved in the response to pathogens.
• Secreted RNase T2 proteins from Fusarium oxysporum contribute to fungal virulence.
• In yeast, RNase T2 mediates selective autophagy of ribosomes induced by starvation.
• The T2 ribonuclease family is widely conserved and includes enzymes with acidic pH optima and non-base-specific cleavage.
• Dysregulation of RNase T2 activity may contribute to autoimmune and autoinflammatory conditions.
• RNase T2 activity is a potential target for modulating immune responses to RNA therapeutics.
• Understanding RNase T2 function aids in crop protection and fungal pathogenesis research.
Mechanism, Genes and Research Methods
What Happens During ribonuclease T2 activity?
In simple terms: RNase T2 cuts RNA in two steps, leaving a cyclic intermediate that is then opened.
The catalytic mechanism of ribonuclease T2 activity proceeds via a two-stage endonucleolytic cleavage. First, the enzyme cleaves the RNA backbone to form a 2',3'-cyclic phosphate intermediate and a 5'-end dephospho-ribonucleoside-RNA. Second, the cyclic phosphate is hydrolyzed to yield a 3'-end 3'-phospho-ribonucleotide-RNA and a 5'-end dephospho-ribonucleoside-RNA. This activity is non-base-specific and typically operates at acidic pH. In mammals, lysosomal RNase T2 cooperates with PLD exonucleases to generate RNA ligands for TLR7 activation, a key step in innate immune sensing.
Substrate Recognition and Cleavage
In simple terms: RNase T2 binds RNA without caring about the sequence and cuts it into small pieces.
RNase T2 enzymes recognize RNA substrates in a non-base-specific manner, meaning they do not discriminate between different nucleotides. The cleavage occurs endonucleolytically, producing 3'-phosphooligonucleotides and nucleoside 3'-phosphates. This broad specificity allows RNase T2 to degrade a wide range of RNA molecules, including ribosomal RNA and viral RNA. In yeast, RNase T2 mediates selective autophagy of ribosomes induced by starvation, suggesting a role in bulk RNA turnover under stress.
Cellular Localization and Compartmentalization
In simple terms: RNase T2 usually works in acidic compartments like lysosomes or vacuoles.
In eukaryotic cells, RNase T2 activity is often found in acidic compartments such as lysosomes and vacuoles. In mammals, lysosomal RNase T2 is required for the generation of RNA ligands for TLR7, a process that occurs in endolysosomes. In plants and fungi, RNase T2 proteins can be secreted, where they may act on extracellular RNA or contribute to virulence. The acidic pH optimum of these enzymes is consistent with their localization to acidic organelles.
Regulation of RNase T2 Activity
In simple terms: RNase T2 activity is controlled by where it is, what it encounters, and cellular stress signals.
RNase T2 activity is regulated at multiple levels, including gene expression, protein trafficking, and substrate availability. In immune cells, the presence of RNA in endolysosomes triggers RNase T2-mediated degradation and TLR activation. Pseudouridine modification of RNA impairs endolysosomal processing and reduces TLR engagement, thereby modulating RNase T2 activity indirectly. In yeast, starvation induces selective autophagy of ribosomes, a process that requires RNase T2. These examples illustrate that RNase T2 activity is tightly linked to cellular stress and immune signaling pathways.
Key Genes Involved in GO:0033897 ribonuclease T2 activity
The following genes and proteins are directly associated with ribonuclease T2 activity (GO:0033897) or its regulation, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| RNASET2 (human) | Lysosomal endonuclease that generates RNA ligands for TLR7 | Innate immunity, autoimmunity, cancer |
| RNASET2 (mouse) | Restricts TLR13-mediated autoinflammation | Autoinflammatory disease models |
| PLD3/PLD4 (human) | Exonucleases cooperating with RNase T2 to generate TLR7 ligands | Endolysosomal RNA processing |
| TLR7 (human) | RNA-sensing Toll-like receptor activated by RNase T2 products | Antiviral immunity, autoimmunity |
| TLR13 (mouse) | RNA-sensing Toll-like receptor restricted by RNase T2 | Autoinflammation |
| LE (tomato) | T2 ribonuclease involved in pathogen response | Plant immunity |
| FoRnt2 (Fusarium oxysporum) | Secreted RNase T2 contributing to virulence | Fungal pathogenesis |
| FocRnt2 (Fusarium oxysporum f. sp. cubense) | RNase T2 contributing to virulence in banana | Fungal pathogenesis |
| Rny1 (Saccharomyces cerevisiae) | RNase T2 mediating selective autophagy of ribosomes | Starvation response, ribosome quality control |
| RNASET2 (zebrafish) | Ortholog used in developmental studies | Conserved function |
| RNASET2 (Drosophila) | Ortholog involved in RNA degradation | Model organism studies |
| RNASET2 (C. elegans) | Ortholog with roles in RNA turnover | Model organism studies |
| RNASET2 (Arabidopsis) | Plant T2 ribonuclease involved in stress responses | Plant biology |
| RNASET2 (bacteria) | Bacterial T2 ribonucleases like RNase II | Microbial RNA metabolism |
| RNASET2 (virus) | Viral T2-like ribonucleases | Viral pathogenesis |
| RNASET2 (parasite) | T2 ribonucleases in protozoan parasites | Host-pathogen interactions |
| RNASET2 (fungi) | Secreted and intracellular T2 ribonucleases | Fungal virulence and saprophytism |
How Is ribonuclease T2 activity Regulated?
Ribonuclease T2 activity is regulated by cellular localization, substrate availability, and stress signals. In immune cells, RNA delivered to endolysosomes is degraded by RNase T2, and the resulting products activate TLR7. Pseudouridine modification of RNA impairs this processing and reduces TLR engagement, thereby modulating the pathway. In yeast, starvation induces selective autophagy of ribosomes, a process that requires RNase T2. Additionally, the expression of RNase T2 genes can be induced by pathogen infection in plants and during fungal virulence. These regulatory mechanisms ensure that RNase T2 activity is deployed appropriately in response to environmental and developmental cues.
ribonuclease T2 activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| RNASET2 (human) | Autoimmune diseases, RNA sensing | Knockout in immune cell lines, TLR7 reporter assays |
| RNASET2 (mouse) | Autoinflammation | Knockout mice, TLR13 activation studies |
| LE (tomato) | Plant pathogen response | Knockout or overexpression in tomato, pathogen infection |
| FoRnt2 (Fusarium oxysporum) | Fungal virulence | Knockout in Fusarium, plant infection assays |
| FocRnt2 (Fusarium oxysporum f. sp. cubense) | Fungal virulence in banana | Knockout in Fusarium, banana infection |
RNase T2 in Autoimmune and Autoinflammatory Diseases
RNase T2 activity is critical for preventing aberrant immune activation by RNA. In mice, loss of RNase T2 leads to TLR13-mediated autoinflammation, demonstrating its role as a negative regulator of RNA-sensing pathways. In humans, lysosomal RNase T2 cooperates with PLD exonucleases to generate RNA ligands for TLR7, and dysregulation of this process can contribute to autoimmune diseases such as systemic lupus erythematosus. Furthermore, pseudouridine-modified RNA evades immune detection by impairing endolysosomal processing and TLR engagement, a mechanism that may be exploited by pathogens or in RNA therapeutics.
RNase T2 in Cancer
Alterations in RNase T2 activity have been linked to cancer biology. Although the exact mechanisms are still being elucidated, RNase T2 is involved in RNA turnover and immune surveillance, processes that can influence tumor progression. The generation of RNA ligands for TLR7 by RNase T2 may affect the tumor microenvironment and antitumor immunity. Further research is needed to fully understand the role of RNase T2 in cancer.
RNase T2 in Plant and Fungal Pathogenesis
In plants, T2 ribonucleases such as tomato LE are involved in the response to pathogens, contributing to defense mechanisms. In fungi, secreted RNase T2 proteins like FoRnt2 and FocRnt2 contribute to virulence in Fusarium oxysporum, highlighting their importance in plant disease. These findings suggest that RNase T2 activity can be a target for crop protection strategies.
From ribonuclease T2 activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does RNase T2 restrict TLR13-mediated autoinflammation? | RNASET2 knockout mouse |
| How does RNase T2 generate TLR7 ligands? | RNASET2 knockout human cell lines, PLD3/4 knockout |
| Does pseudouridine modification affect RNase T2 processing? | In vitro RNA degradation assays with modified RNA |
| What is the role of RNase T2 in plant immunity? | Tomato LE knockout or overexpression lines |
| How does fungal RNase T2 contribute to virulence? | FoRnt2 or FocRnt2 knockout in Fusarium, plant infection |
| Is RNase T2 required for ribosome autophagy? | Yeast Rny1 knockout, starvation conditions |
How to Study the ribonuclease T2 activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Global RNA levels and transcript stability | Assessing RNase T2 impact on transcriptome |
| PARE/degradome | Cleavage sites and endonucleolytic activity | Identifying RNase T2 substrates |
| In vitro RNase assay | Enzymatic activity and kinetics | Characterizing purified RNase T2 |
| Fluorescence microscopy | Subcellular localization | Co-localization with lysosomal markers |
| TLR reporter assay | Immune activation by RNA ligands | Measuring TLR7/TLR13 signaling |
| Cytokine ELISA | Cytokine production | Quantifying immune responses |
| Yeast starvation assay | Ribosome autophagy | Studying RNase T2 in stress responses |
| Pathogen infection assay | Virulence and plant defense | Testing fungal RNase T2 mutants |
RNA Sequencing and Degradome Analysis
RNA-seq can be used to assess global changes in RNA levels upon modulation of RNase T2 activity. Degradome analysis, such as parallel analysis of RNA ends (PARE), can identify cleavage sites and confirm the endonucleolytic activity of RNase T2 in vivo. These methods are particularly useful for understanding substrate specificity and the impact of RNase T2 on transcriptomes.
Biochemical Assays for RNase T2 Activity
In vitro ribonuclease assays using synthetic RNA substrates can measure the catalytic activity of purified RNase T2 enzymes. These assays typically monitor the formation of 2',3'-cyclic phosphate intermediates or the release of acid-soluble nucleotides. Such assays are essential for characterizing enzyme kinetics, pH optima, and inhibitor sensitivity.
Imaging and Localization Studies
Fluorescence microscopy with tagged RNase T2 proteins can reveal their subcellular localization, such as lysosomes or vacuoles. Co-localization with markers like LAMP1 or TLR7 can provide insights into functional compartments. Live-cell imaging can track the trafficking of RNase T2 and its substrates.
Immune Activation Assays
Reporter cell lines expressing TLR7 or TLR13 can be used to measure immune activation by RNA degradation products generated by RNase T2. These assays are valuable for studying the role of RNase T2 in innate immunity and autoinflammation. Cytokine production can be quantified by ELISA or luciferase reporters.
How CRISPR Can Be Used to Study GO:0033897 ribonuclease T2 activity
Knockout
CRISPR knockout of RNase T2 genes (e.g., RNASET2 in human cells or Rny1 in yeast) can abolish enzyme activity, allowing researchers to study loss-of-function phenotypes such as impaired TLR7 ligand generation or defective ribosome autophagy. Knockout models are essential for validating the role of RNase T2 in immune sensing and cellular stress responses.
Point Mutation
Introducing point mutations in the catalytic residues of RNase T2 can dissect the enzymatic mechanism and separate catalytic activity from non-catalytic functions. For example, mutations in the active site histidines can abolish ribonuclease activity while preserving protein interactions. Such models are valuable for understanding structure-function relationships.
Knock-in
Knock-in of tagged RNase T2 (e.g., GFP or HA) allows for precise localization and interaction studies. Tagged knock-in models can be used to track endogenous protein expression and trafficking in real time. Additionally, knock-in of disease-associated mutations can model human pathologies.
Overexpression
Overexpression of RNase T2 can enhance RNA degradation and immune activation, providing a gain-of-function system to study downstream effects. For instance, overexpression in plant or fungal cells can increase virulence or trigger defense responses. Overexpression models are also useful for producing recombinant enzyme for biochemical assays.
How EDITGENE Supports ribonuclease T2 activity Research
Researchers studying ribonuclease T2 activity-related genes often need to determine whether a candidate gene is causally involved in RNA processing, immune sensing, or pathogenesis. This requires precise genetic manipulation and functional validation. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate such research.
Contact EDITGENE today to design your custom CRISPR model for ribonuclease T2 activity research.
Frequently Asked Questions About ribonuclease T2 activity
What is ribonuclease T2 activity?
Ribonuclease T2 activity (GO:0033897) is a molecular function that catalyzes the two-stage endonucleolytic cleavage of RNA to nucleoside 3'-phosphates and 3'-phosphooligonucleotides via 2',3'-cyclic phosphate intermediates.
What genes are involved in ribonuclease T2 activity?
Key genes include RNASET2 in humans and mice, PLD3 and PLD4, TLR7, TLR13, tomato LE, Fusarium FoRnt2 and FocRnt2, and yeast Rny1.
How does RNase T2 activate TLR7?
Lysosomal RNase T2 cooperates with PLD exonucleases to degrade RNA into ligands that activate TLR7, a process important for innate immune sensing.
What diseases are associated with RNase T2 dysfunction?
Dysfunction of RNase T2 is linked to autoinflammatory conditions via TLR13, autoimmune diseases through TLR7, and potentially cancer and plant/fungal pathogenesis.
Is RNase T2 activity base-specific?
No, RNase T2 activity is non-base-specific, meaning it cleaves RNA regardless of nucleotide sequence.
What is the pH optimum of RNase T2?
RNase T2 enzymes typically have an acidic pH optimum, consistent with their localization in lysosomes and vacuoles.
How can I study RNase T2 activity in the lab?
Common methods include RNA-seq, degradome analysis, in vitro ribonuclease assays, fluorescence microscopy, and TLR reporter assays.
What CRISPR models are available for RNase T2 research?
Knockout, point mutation, knock-in, and overexpression models can be generated for RNase T2 genes to study loss- and gain-of-function phenotypes.
Does RNase T2 play a role in autophagy?
Yes, in yeast, RNase T2 (Rny1) mediates selective autophagy of ribosomes induced by starvation.
How does pseudouridine modification affect RNase T2?
Pseudouridine-modified RNA avoids immune detection through impaired endolysosomal processing and reduced TLR engagement, a process that involves RNase T2.
Conclusion
Ribonuclease T2 activity (GO:0033897) is a fundamental molecular function with critical roles in RNA turnover, innate immune sensing, and cellular stress responses. Its involvement in TLR7 and TLR13 pathways, plant and fungal pathogenesis, and ribosome autophagy underscores its broad biological importance. Understanding the mechanisms and regulation of RNase T2 activity can provide insights into autoimmune diseases, host-pathogen interactions, and RNA metabolism. EDITGENE offers comprehensive CRISPR services to facilitate functional studies of RNase T2 and its related genes, empowering researchers to uncover new therapeutic targets and biological insights.
References
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- 2. Bérouti M et al.. 2025. Pseudouridine RNA avoids immune detection through impaired endolysosomal processing and TLR engagement.. Cell 188(18):4880-4895.e15 PMID: 40580950
- 3. Singh NK et al.. 2020. Tomato T2 ribonuclease LE is involved in the response to pathogens.. Mol Plant Pathol 21(7):895-906 PMID: 32352631
- 4. Gomez-Diaz C et al.. 2025. RNase T2 restricts TLR13-mediated autoinflammation in vivo.. J Exp Med 222(3) PMID: 39853306
- 5. Qian H et al.. 2022. The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence.. Mol Plant Pathol 23(9):1346-1360 PMID: 35696123
- 6. He Y et al.. 2024. A ribonuclease T2 protein FocRnt2 contributes to the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.. Mol Plant Pathol 25(8):e13502 PMID: 39118198
- 7. Deshpande RA et al.. 2002. Ribonucleases from T2 family.. Crit Rev Microbiol 28(2):79-122 PMID: 12109772
- 8. Minami A et al.. 2025. The ribonuclease RNase T2 mediates selective autophagy of ribosomes induced by starvation in Saccharomyces cerevisiae.. J Biol Chem 301(6):108554 PMID: 40294649
