TSEN15 Antibody

What is TSEN15 and why is it important in cellular function?

TSEN15 (tRNA Splicing Endonuclease 15) functions as a structural protein within the heterotetrameric tRNA splicing endonuclease complex. This complex is responsible for removing introns from precursor tRNAs, a critical step in tRNA maturation. The TSEN complex consists of four subunits: TSEN2 and TSEN34 (catalytic subunits) that form the active site, and TSEN54 and TSEN15 (structural proteins) . While not directly catalytic, TSEN15 is essential for maintaining proper complex assembly and function. Mutations in TSEN15 have been linked to neurodevelopmental disorders, particularly a milder form of pontocerebellar hypoplasia type 2 (PCH2) characterized by progressive microcephaly and global developmental delay . The protein interacts specifically with TSEN34 to form a stable heterodimer that subsequently assembles with the TSEN2-54 heterodimer to create the functional tetrameric complex .

How does TSEN15 interact with other components of the tRNA splicing machinery?

TSEN15 forms a stable heterodimer with TSEN34 through conserved nuclease fold interactions . This heterodimer then associates with the TSEN2-54 heterodimer to form the complete tetrameric complex. The assembly occurs in a stepwise manner:

• TSEN15 binds to TSEN34, forming a non-catalytic heterodimer

• TSEN2 binds to TSEN54, forming another heterodimer

• These two heterodimers combine to form the active tetrameric endonuclease

This assembly process occurs independently of CLP1 (a kinase that associates with TSEN) and does not require ATP . The interactions between subunits are specific and strong, as demonstrated by co-immunoprecipitation experiments showing that TSEN15 can pull down other TSEN subunits when overexpressed . Importantly, the nuclease fold observed in the TSEN15-34 X-ray crystal structure is evolutionarily conserved, suggesting similar interaction patterns throughout the complex .

What validation methods should be used for TSEN15 antibodies?

When validating TSEN15 antibodies for research, consider these essential approaches:

• Western blot analysis: Verify specificity using recombinant TSEN15 protein as a positive control and TSEN15-knockout cell lines as negative controls. The expected molecular weight of human TSEN15 is approximately 19 kDa .

• Immunoprecipitation: Confirm antibody functionality by performing pull-down experiments. A properly functioning TSEN15 antibody should co-precipitate other TSEN complex members (TSEN2, TSEN34, and TSEN54) .

• Subcellular localization: Use immunofluorescence to verify the predominantly nuclear localization of TSEN15, consistent with its role in pre-tRNA processing.

• Cross-reactivity testing: Ensure specificity by testing against other TSEN family members, particularly in overexpression systems.

• Application-specific validation: For specialized applications like ChIP or flow cytometry, perform application-specific validation using appropriate controls.

What are the typical applications for TSEN15 antibodies in research?

TSEN15 antibodies serve multiple research purposes:

• Protein expression analysis: Western blotting to detect TSEN15 levels in various tissues or under different conditions .

• Complex assembly studies: Co-immunoprecipitation to investigate how TSEN15 interacts with other TSEN subunits and how mutations affect these interactions .

• Subcellular localization: Immunofluorescence microscopy to determine where TSEN15 functions within cells.

• Biomarker research: TSEN15 has been identified as part of a three-gene diagnostic model for acute rejection in renal transplant patients, making it potentially useful in transplantation research .

• Disease mechanism studies: Investigating how mutations in TSEN15 contribute to neurodevelopmental disorders by affecting protein stability, complex formation, or enzymatic activity .

How do disease-associated mutations in TSEN15 affect protein function and complex assembly?

TSEN15 mutations associated with pontocerebellar hypoplasia (PCH) affect protein function through multiple mechanisms:

• Protein stability: The p.Trp76Gly and p.Tyr152Cys mutations significantly reduce TSEN15 protein abundance in expression systems, with p.Trp76Gly having a more severe effect. In contrast, the p.His116Tyr mutation does not affect protein levels .

• Complex stoichiometry: All three reported pathogenic mutations (p.His116Tyr, p.Trp76Gly, and p.Tyr152Cys) alter the stoichiometry of the TSEN complex. The p.His116Tyr mutation decreases association with partner proteins, while p.Trp76Gly and p.Tyr152Cys paradoxically increase partner association relative to TSEN15 levels .

• Enzymatic activity: Purified complexes containing TSEN15 mutations show compromised pre-tRNA cleavage activity in vitro, though the effect in vivo may be less severe, explaining the milder phenotype compared to other PCH-causing mutations .

• Thermal stability: The p.His116Tyr mutation thermally destabilizes the entire endonuclease complex, potentially affecting its function under cellular stress conditions .

These findings suggest that TSEN15 mutations don't completely abolish complex formation but rather subtly alter interactions between subunits, resulting in reduced functionality that particularly affects neuronal development.

What experimental approaches can be used to study TSEN15-dependent pre-tRNA processing?

To investigate TSEN15's role in pre-tRNA processing:

• In vitro cleavage assays: Purify recombinant TSEN complexes with wild-type or mutant TSEN15 and assess their ability to cleave labeled pre-tRNA substrates. Use yeast pre-tRNA^Phe^ as a model substrate, as it has been validated for human TSEN activity .

• Active site mutation analysis: Create TSEN complexes with mutations in the catalytic subunits (TSEN2-H377A or TSEN34-H255A) alongside wild-type or mutant TSEN15 to distinguish between effects on complex assembly versus catalytic activity .

• Cellular pre-tRNA analysis: Quantify pre-tRNA and mature tRNA levels in cells expressing mutant TSEN15 using northern blotting or quantitative RT-PCR to assess processing defects.

• tRNA-sequencing: Perform high-throughput sequencing of tRNAs to comprehensively profile the impact of TSEN15 mutations on the tRNA pool.

• Patient-derived models: Establish fibroblast or induced pluripotent stem cell models from patients with TSEN15 mutations to study pre-tRNA processing defects in a disease-relevant context .

How can TSEN15 antibodies be used to investigate the relationship between tRNA processing and neurodevelopmental disorders?

TSEN15 antibodies enable several approaches to study the link between tRNA processing and neurodevelopmental disorders:

• Expression profiling in neural tissues: Use antibodies to compare TSEN15 expression levels and localization in different brain regions and during developmental stages to identify vulnerable cell populations.

• Mutation impact analysis: Employ co-immunoprecipitation with TSEN15 antibodies to assess how PCH-causing mutations affect complex assembly in patient-derived neural cells .

• Substrate binding studies: Combine TSEN15 antibodies with RNA immunoprecipitation to identify which pre-tRNAs are most affected by TSEN15 mutations.

• Developmental timing studies: Track TSEN15 expression and complex formation during critical neurodevelopmental windows to determine when tRNA processing defects first manifest.

• Compensatory mechanism investigation: Use TSEN15 antibodies to identify potential protein interactors that might compensate for reduced TSEN activity in less vulnerable cell types.

What methods are most effective for analyzing TSEN15 in functional genomics studies?

For functional genomics investigations of TSEN15:

• CRISPR-Cas9 mutagenesis: Generate cell lines with specific TSEN15 mutations or conditional knockouts to study phenotypic effects. Complete knockout may be lethal due to TSEN15's essential function .

• RNA-Seq analysis: Assess global transcriptome changes in cells with TSEN15 mutations, focusing on pathways affected by altered tRNA processing.

• Proteomics approach: Combine TSEN15 antibody-mediated immunoprecipitation with mass spectrometry to identify the complete interaction network of TSEN15 under normal and disease conditions.

• Single-cell analysis: Use TSEN15 antibodies in single-cell proteomics approaches to understand cell-type-specific vulnerabilities to TSEN15 dysfunction.

• Integrative genomics: Correlate TSEN15 expression or mutation data with phenotypic outcomes in large datasets to identify genetic modifiers of TSEN15-related disease severity.

Table 1: Comparison of TSEN15 Mutations and Their Effects on Complex Assembly and Function

How can the TSEN15 expression level be reliably quantified in patient samples?

Accurate quantification of TSEN15 in patient samples requires multiple complementary approaches:

• Quantitative western blotting: Use TSEN15 antibodies with reliable loading controls and standard curves of recombinant protein to determine absolute protein levels in patient-derived cells or tissues .

• RT-qPCR: Measure TSEN15 mRNA expression levels with carefully validated primers and reference genes appropriate for the tissue being studied.

• Digital droplet PCR: For more sensitive and absolute quantification of TSEN15 transcript levels, especially in limited samples.

• Proximity ligation assay: Combine TSEN15 antibodies with antibodies against other TSEN components to quantify not just expression but also complex formation in situ.

• Mass spectrometry: Use targeted proteomics approaches for absolute quantification of TSEN15 protein in complex samples.

For diagnostic applications, TSEN15 expression has shown promising results as part of a three-gene diagnostic model (with CAPRIN1 and PRR34-AS1) for acute rejection in renal transplant patients, achieving high accuracy (AUC = 0.968 in training cohort, AUC = 0.925 in validation cohort) .

What considerations are important when using TSEN15 as part of a diagnostic biomarker panel?

When developing or using TSEN15 as a biomarker:

• Assay standardization: Ensure consistent antibody performance across batches and laboratories using standard reference materials.

• Context-specific validation: Although TSEN15 shows promise in transplant rejection diagnostics , its utility must be validated in each specific clinical context.

• Sample type considerations: Determine optimal sample types (whole blood, specific cell fractions, tissues) for TSEN15 assessment based on expression patterns.

• Combinatorial approach: As demonstrated in transplant rejection studies, TSEN15 performs best as part of a multi-gene panel (with CAPRIN1 and PRR34-AS1) rather than in isolation .

• Technical platform selection: Consider the strengths and limitations of different detection platforms (qPCR, NanoString, antibody-based assays) for clinical implementation.

• Reference ranges: Establish normal reference ranges for TSEN15 expression across relevant populations and disease states.

What are the optimal conditions for using TSEN15 antibodies in co-immunoprecipitation experiments?

For successful TSEN15 co-immunoprecipitation:

• Lysis conditions: Use mild lysis buffers containing 0.5% NP-40 or 1% Triton X-100 with 150mM NaCl to preserve complex integrity. Avoid harsh detergents like SDS that may disrupt protein-protein interactions .

• Antibody selection: Choose antibodies targeting the C-terminal region of TSEN15, as this region is less likely to be involved in interactions with other TSEN components .

• Controls: Include appropriate controls such as IgG isotype controls and, ideally, TSEN15-knockout cell extracts.

• Affinity tag alternatives: Consider using tagged TSEN15 (e.g., His-Flag-tagged TSEN15 or SBP-TEV-TSEN15) for cleaner pull-downs in recombinant systems .

• Complex elution: To maintain complex integrity, use gentle elution methods such as peptide competition rather than harsh denaturation.

• Crosslinking considerations: For transient interactions, consider using reversible crosslinking approaches to stabilize the complexes.

How can researchers effectively study the impact of TSEN15 mutations on complex assembly?

To investigate how mutations affect TSEN complex assembly:

• Heterologous expression systems: Express wild-type or mutant TSEN15 along with other complex components in HEK293 cells or insect cells for biochemical analysis .

• Tandem affinity purification: Use sequential purification steps with tagged TSEN15 to isolate pure complexes for analysis of subunit stoichiometry .

• Size exclusion chromatography: Analyze complex formation and stability using gel filtration to separate assembled complexes from individual components or subcomplexes .

• Thermal shift assays: Assess the impact of mutations on complex stability through thermal denaturation experiments .

• Native gel electrophoresis: Use blue native PAGE to analyze intact complexes and detect shifts in complex size or composition.

• Quantitative mass spectrometry: Apply SILAC or TMT labeling approaches to precisely quantify changes in subunit stoichiometry caused by mutations.

What strategies can be employed to enhance the specificity of TSEN15 antibodies in complex biological samples?

To improve TSEN15 antibody specificity:

• Pre-adsorption: Incubate antibodies with recombinant TSEN15 protein to remove non-specific antibodies from polyclonal preparations.

• Epitope selection: Choose antibodies targeting unique regions of TSEN15 with minimal homology to other proteins, particularly other TSEN family members.

• Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers) to minimize background in immunoblotting applications.

• Knockout validation: Validate antibody specificity using CRISPR-edited cell lines lacking TSEN15 expression.

• Competitive blocking: In tissue samples with high background, perform parallel experiments with excess unlabeled antibody to identify non-specific signals.

• Antibody concentrations: Determine optimal antibody dilutions for each sample type and application through careful titration experiments.