TSSC4 Human
Description
Introduction to TSSC4 Human
TSSC4 (tumor suppressing subtransferable candidate 4) is a 36.8 kDa intracellular protein encoded by the TSSC4 gene located on chromosome 11p15.5, within an imprinted region containing other tumor suppressors like CDKN1C and PHLDA2 . Initially identified as a tumor suppressor, TSSC4 has emerged as a multifunctional protein with roles in RNA splicing, autophagy regulation, and cancer biology. A single-nucleotide polymorphism (SNP) in TSSC4 is associated with reduced human height, highlighting its evolutionary significance .
Domain Architecture and Interactions
TSSC4 exhibits an intrinsically disordered structure with six conserved homology regions (Hom1–Hom6) . Key functional domains include:
Key Insights:
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Hom2 and Hom4 are essential for TSSC4’s role in U5 snRNP maturation and tri-snRNP assembly .
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The LIR domain (aa 97–100) is critical for inhibiting autophagy by blocking LC3 lipidation .
Mechanism of Action
TSSC4 acts as a chaperone in U5 snRNP biogenesis and post-splicing recycling:
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Tri-snRNP Formation: Promotes assembly of U4/U6- U5 tri-snRNP by facilitating U5 snRNP interaction with U4/U6 di-snRNP .
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Post-Splicing Recycling: Associates with the U5/PRPF19 complex, enabling regeneration of functional U5 snRNP for subsequent splicing cycles .
Splicing Defects Upon TSSC4 Depletion
| Splicing Event Type | Number of Affected Events | Example Genes |
|---|---|---|
| Alternative Exon Usage | 689 total events | CCND1, PCNA |
| Intron Retention | 145 events | – |
| Cassette Exon | 89 events | – |
| Mutually Exclusive Exons | 74 events | – |
Key Data:
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TSSC4 knockout in HeLa cells disrupts splicing of 1,316 genes, including oncogenes like CCND1 .
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Defects in tri-snRNP stability are observed without changes in core U5 snRNP composition .
Tumor Suppression and Autophagy Inhibition
Mechanistic Links:
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Autophagy Suppression: Binds LC3-I/II via its LIR domain, preventing autophagosome formation .
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EGFR Signaling: Upregulated by EGFR activation in GBM, linking growth signaling to autophagy inhibition .
Experimental Evidence
| Cell Line | TSSC4 Overexpression | LC3B-II Levels (CQ-treated) |
|---|---|---|
| U87 (GBM) | Wild-type | 0.7 (basal), 1.3 (TMZ-induced) |
| MDA-MB-231 | Wild-type | 50% reduction in autophagy flux |
| U373 | TSSC4M (mutant) | No inhibition |
Key Findings:
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TSSC4 overexpression reduces autophagy by 50% in tumorspheres, limiting cancer stem cell survival .
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TSSC4M (LIR mutant) fails to inhibit autophagy, confirming LC3 interaction dependency .
Diagnostic and Therapeutic Targets
Future Directions:
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Targeting TSSC4 in cancers with low autophagy flux (e.g., glioblastoma).
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Exploring TSSC4’s role in neurodegenerative diseases linked to splicing defects.
Product Specs
Q&A
What is the primary function of TSSC4 in human cells?
TSSC4 serves as a component of U5 snRNP that promotes tri-snRNP formation. It functions as a specific chaperone that acts in both U5 snRNP de novo biogenesis and post-splicing recycling. As an intrinsically disordered protein, TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites . This binding inhibits SNRNP200 helicase activity and spatially aligns proteins, which coordinates the formation of a U5 sub-module while transiently blocking premature interaction of SNRNP200 with other spliceosomal factors .
Which specific protein interactions does TSSC4 participate in within the spliceosome?
TSSC4 interacts with several U5-specific proteins, primarily:
| Protein Partner | Interaction Region in TSSC4 | Functional Significance |
|---|---|---|
| PRPF8 | Conserved binding regions | Coordinates U5 snRNP assembly |
| SNRNP200 | Non-contiguous binding sites | Inhibits helicase activity |
| EFTUD2 | Multiple regions | Supports tri-snRNP formation |
Additionally, TSSC4 associates with U5 snRNP chaperones and the PRPF19 complex, forming distinct domains that are critical for interactions with U5 snRNP and the PRPF19 complex . These interactions are essential for TSSC4's function in tri-snRNP assembly.
How is TSSC4 structurally characterized and what methodologies are used to study its intrinsic disorder?
TSSC4 is characterized as an intrinsically disordered protein that employs short, discontinuous, and conserved binding regions to interact with its partners. Researchers use multiple complementary approaches to study its structure and interactions:
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Limited proteolysis: Mixtures of SNRNP200 HR and GST-TSSC4 are treated with chymotrypsin, and reaction products are analyzed via analytical size exclusion chromatography (SEC) .
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Peptide SPOT arrays: Membranes with spots of 25-residue peptides of TSSC4 with an overlap of 20 residues are used to identify specific binding regions .
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Cryogenic electron microscopy (cryoEM): This technique provides high-resolution structural analysis of TSSC4 in complex with the helicase region of SNRNP200 and the Jab1/MPN domain of PRPF8 .
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Protein reconstitution assays: His-tagged SNRNP200 and GST-tagged TSSC4 are produced separately, mixed, captured on resin, and analyzed by SEC to study complex formation .
What experimental approaches can effectively examine TSSC4's role in promoting tri-snRNP formation?
To investigate TSSC4's role in tri-snRNP formation, researchers should implement a multi-faceted approach:
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TSSC4 Depletion Studies: siRNA-mediated knock-down of TSSC4 can be performed, followed by analysis of U4, U5, and U6 snRNA accumulation in Cajal bodies and measurement of U4/U6-U5 tri-snRNP levels versus free U5 snRNP .
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Domain Mapping: Based on structural insights, researchers can design TSSC4 variants that lack stable binding to the PRPF8 Jab1/MPN domain or SNRNP200. These variants can be employed in comparative protein and RNA interactome studies using immunoprecipitation/mass spectrometry from nuclear extracts .
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Competitive Binding Assays: To understand how TSSC4 might displace other factors, researchers can use C-terminal fragments of proteins like FBP21 (residues 200-376) in competitive binding assays with SNRNP200 HR complexes .
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RNA Helicase Activity Assays: Since TSSC4 inhibits SNRNP200 helicase activity, assays measuring this activity in the presence and absence of TSSC4 or its variants can provide functional insights.
How can researchers effectively analyze the TSSC4-mediated inhibition of SNRNP200 helicase activity?
To analyze TSSC4's inhibition of SNRNP200 helicase activity, researchers should consider these methodological approaches:
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In vitro Helicase Assays: Using purified recombinant SNRNP200 and TSSC4 proteins, researchers can measure RNA unwinding activity with fluorescently labeled RNA duplexes in the presence and absence of TSSC4.
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Structure-Based Mutagenesis: Based on the cryoEM structure of the TSSC4-SNRNP200-PRPF8 complex, researchers can design mutations in the TSSC4 binding interface with SNRNP200 to identify residues critical for helicase inhibition .
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ATP Hydrolysis Assays: Since helicase activity is coupled to ATP hydrolysis, measuring ATP consumption rates by SNRNP200 in the presence of different concentrations of TSSC4 can quantify inhibition efficiency.
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Single-Molecule Techniques: Techniques such as FRET (Förster Resonance Energy Transfer) can be employed to observe the dynamics of SNRNP200-mediated RNA unwinding and how TSSC4 affects these dynamics at the single-molecule level.
What strategies can be employed to investigate TSSC4's role in post-splicing U5 snRNP recycling?
TSSC4 participates not only in de novo U5 snRNP biogenesis but also in post-splicing recycling. To investigate this role:
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Pulse-Chase Experiments: Using metabolic labeling of newly synthesized RNA, researchers can track the fate of U5 snRNA after splicing and determine how TSSC4 depletion affects recycling rates.
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Immunoprecipitation of Post-Splicing Complexes: Researchers can immunoprecipitate U5/PRPF19 post-splicing particles and analyze TSSC4 association during the recycling process .
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Live Cell Imaging: Using fluorescently tagged TSSC4 and components of the post-splicing complex, researchers can visualize their co-localization and dynamics in living cells.
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In vitro Reconstitution of Recycling: Purified post-splicing U5 particles can be incubated with recombinant TSSC4 and other recycling factors to reconstitute and study the recycling process biochemically.
How can researchers resolve contradicting data on TSSC4's interaction with the spliceosome assembly pathway?
When encountering contradictory data regarding TSSC4's interactions:
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Cell Type-Specific Analysis: Examine TSSC4 interactions across different cell types, as splicing regulation can vary between tissues.
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Environmental Condition Variables: Test interactions under different cellular stresses, as TSSC4's function might be modulated by cellular conditions.
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Isoform-Specific Studies: Analyze whether alternative TSSC4 isoforms might exhibit different interaction profiles.
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Temporal Analysis: Examine TSSC4 interactions at different time points during splicing to capture dynamic associations that might be missed in endpoint analyses.
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Methodology Comparison: When contradictory results emerge, systematically compare the methodologies used. For example, antibody-based techniques versus tagged protein approaches, or in vitro versus in vivo studies, may yield different results due to methodological limitations .
What protein production systems are optimal for studying TSSC4 and its binding partners?
For optimal production of TSSC4 and its binding partners:
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Expression Systems: E. coli expression using the pETM30 vector system has been successfully employed for producing N-terminally His6-GST-tagged TSSC4 (TEV-cleavable) .
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Purification Strategy:
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Glutathione Sepharose affinity chromatography for initial capture
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TEV protease treatment for tag removal
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Size exclusion chromatography for final purification
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Complex Reconstitution: For TSSC4-SNRNP200 complexes, mixing individually produced His-SNRNP200 HR and GST-TSSC4, followed by affinity capture and SEC has proven effective .
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Storage Conditions: Purified proteins and complexes can be concentrated to ~5.7 mg/ml, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C for long-term stability .
What detection methods are most effective for studying TSSC4's interactions in human cells?
For studying TSSC4's interactions in human cells:
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Immunoprecipitation/Mass Spectrometry: This approach can reveal distinct interaction profiles of wild-type TSSC4 and binding-deficient variants with snRNP proteins, other spliceosomal proteins, and chaperones .
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Western Blotting: Commercial antibodies against SNRNP200 (such as anti-SNRNP200 antibody produced in rabbit) can be used at concentrations of 0.04-0.4 μg/mL for immunoblotting .
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Immunofluorescence: For cellular localization studies, antibodies can be used at 0.25-2 μg/mL to visualize TSSC4 and its interacting partners in situ .
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Immunohistochemistry: For tissue sections, antibody dilutions of 1:200-1:500 are recommended for optimal staining .
How might TSSC4 function be modulated in disease states affecting splicing mechanisms?
Given TSSC4's integral role in spliceosome assembly and recycling, investigating its function in splicing-related diseases presents an important research direction. Researchers should consider:
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Expression Analysis: Examining TSSC4 expression levels in tissues from patients with splicing-related diseases compared to healthy controls.
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Mutation Screening: Sequencing TSSC4 in patient cohorts to identify potentially pathogenic variants that might affect its interaction with PRPF8, SNRNP200, or other partners.
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Functional Assays: Developing reporter assays to measure how disease-associated variants affect TSSC4's ability to promote tri-snRNP formation and snRNP recycling.
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Therapeutic Potential: Exploring whether modulating TSSC4 function might restore normal splicing in diseases characterized by splicing defects.
What is the evolutionary conservation of TSSC4 function across species, and how can comparative studies enhance our understanding?
Understanding TSSC4's evolutionary history can provide insights into its core functions:
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Sequence Analysis: Comparing TSSC4 sequences across species to identify conserved regions that likely represent functionally critical domains.
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Functional Complementation: Testing whether TSSC4 from different species can complement TSSC4 depletion in human cells.
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Structure Comparison: Analyzing whether the intrinsically disordered nature of TSSC4 is conserved across species and how this relates to function.
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Interaction Conservation: Determining whether TSSC4's interactions with PRPF8, SNRNP200, and EFTUD2 are conserved in orthologous proteins from other species.
Product Science Overview
TSSC4 has been identified as a novel tumor suppressor and autophagy inhibitor. Autophagy is a cellular process involving the degradation and recycling of cellular components, which can play a dual role in cancer by either promoting cell survival or inducing cell death . In the context of glioblastoma (GBM), the most aggressive type of brain cancer in adults, TSSC4 has shown significant potential in inhibiting cancer cell growth .
TSSC4 interacts with the autophagy protein LC3, inhibiting autophagy-induced cell death (AuICD) in cancer cells . This interaction is crucial for its tumor-suppressing function. Overexpression of TSSC4 has been shown to prevent cell death induced by temozolomide (TMZ), a standard chemotherapy drug used in GBM treatment . When TSSC4 is mutated in its conserved LC3-interacting region, it loses its ability to inhibit autophagy and prevent cell death .
Recent studies have demonstrated that TSSC4 expression is upregulated by the activation of the epidermal growth factor receptor (EGFR) in GBM cells . EGFR variant III (EGFRvIII) is the most common mutation in GBM, and its expression leads to increased levels of TSSC4, which in turn decreases TMZ-induced cell death . Knockout of TSSC4 in EGFRvIII-expressing GBM cells results in increased autophagy and cell death, suggesting that TSSC4 downregulation promotes TMZ-induced AuICD .
