Recombinant Mouse tRNA-splicing endonuclease subunit Sen34 (Tsen34)

What is the primary role of TSEN34 in RNA processing?

TSEN34 is a catalytically active subunit of the tRNA splicing endonuclease (TSEN) complex that plays a critical role in pre-tRNA maturation. Its primary known function is to catalyze the cleavage at the 3' splice site of intron-containing pre-tRNAs during the splicing process. This activity is essential for generating mature, functional tRNAs that can participate in protein synthesis. Recent cryo-EM structures have confirmed that TSEN34 is positioned surrounding the 3' splice site in pre-tRNA–bound complexes, where it performs its endonucleolytic function . The catalytic mechanism involves a catalytic triad of residues that coordinate the cleavage reaction, with structural studies revealing that R279 and W306 of TSEN34 are positioned similarly to the arginine tweezer pair found in archaeal endonucleases .

What interactions does TSEN34 form within the TSEN complex?

TSEN34 engages in extensive interactions with all other subunits in the TSEN complex. It forms a particularly intricate interface with TSEN54, involving hydrogen bonds, salt bridges, and hydrophobic interactions with a contact surface area of approximately 2287.2 Ų . This association is critical for positioning the mature domain of pre-tRNAs through polar contacts with the phosphoribose backbone of the acceptor stem, D-stem, and anticodon stem. The extensive TSEN34–54 contact surface to the pre-tRNA covers an area of approximately 1838.3 Ų, explaining why the complex can bind mature tRNAs with similar dissociation constants as pre-tRNAs . Additionally, TSEN34 interacts with TSEN2 and TSEN15, collectively forming the functional architecture required for accurate tRNA processing.

What is known about TSEN34 conservation across species?

TSEN34 is highly conserved across eukaryotes, reflecting the essential nature of tRNA splicing. Structural and bioinformatic analyses indicate that orthologs from yeast, human, and Drosophila share considerable sequence and structural similarity, particularly in the catalytic domains. AlphaFold structure predictions for TSEN34 orthologs show consistent structural features, including the characteristic insertion between N and C subdomains . The conservation of TSEN homologs throughout eukaryotes and some archaea suggests that the tRNA splicing function is evolutionarily ancient and preserved. In yeast, the TSEN34 ortholog is known as Sen34, and it functions similarly in the tRNA splicing process, highlighting the fundamental conservation of this RNA processing machinery .

What are the optimal conditions for expressing and purifying recombinant mouse TSEN34?

Based on established protocols for recombinant proteins, mouse TSEN34 is typically expressed in an E. coli expression system using pET-based vectors with an affinity tag (commonly a His-tag) for purification purposes. For optimal expression, induction with IPTG at concentrations between 0.5-1.0 mM when cultures reach OD600 of 0.6-0.8, followed by growth at lower temperatures (16-18°C) overnight, often yields better results than standard conditions. Purification typically involves immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by size exclusion chromatography to achieve high purity.
For storage stability, similar to other recombinant proteins like the described mouse Syndecan-3, TSEN34 should be stored in a suitable buffer (typically PBS with glycerol) at -80°C, avoiding repeated freeze-thaw cycles . When working with the complete TSEN complex, co-expression of all four subunits or reconstitution from individually purified components may be necessary to obtain functionally active complexes for enzymatic or structural studies.

How can researchers assess the catalytic activity of recombinant TSEN34 in vitro?

The catalytic activity of recombinant TSEN34 can be assessed through in vitro cleavage assays using synthesized pre-tRNA substrates. These assays typically involve incubating the purified TSEN complex (or reconstituted complex containing TSEN34) with radiolabeled or fluorescently tagged pre-tRNA substrates, followed by analysis of cleavage products using denaturing PAGE. Since TSEN34 specifically catalyzes cleavage at the 3' splice site, researchers can design substrates with modifications at this site to specifically probe TSEN34 activity.
It's important to note that TSEN34 functions as part of the heterotetrameric complex, and its activity is highly dependent on proper complex formation with other TSEN subunits. Recent structural studies have used pre-tRNAs with specific modifications to prevent cleavage while maintaining binding, such as replacing the 3' and 5' splice site ribose OH groups with fluorine . For detailed kinetic analyses, researchers can measure cleavage rates under varying substrate concentrations, enabling determination of parameters such as Km and kcat values specific to TSEN34-mediated catalysis.

What strategies can be employed to study TSEN34 interactions with RNA substrates?

Multiple complementary approaches can be used to study TSEN34-RNA interactions:

• Cryo-EM analysis has proven highly effective in visualizing TSEN34 interactions with pre-tRNA substrates. Recent studies achieved resolutions of 3.1-3.8Å for the pre-tRNA-bound complex, revealing specific contacts between TSEN34 and the RNA substrate .

• UV crosslinking experiments combined with mass spectrometry can identify specific amino acid residues that contact the RNA. This approach involves irradiating the protein-RNA complex with UV light to create covalent bonds at the interaction sites.

• Electrophoretic mobility shift assays (EMSAs) can be used to determine binding affinities between TSEN34 (or the complete TSEN complex) and various RNA substrates, including both canonical pre-tRNAs and potential non-canonical substrates.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into conformational changes in TSEN34 upon RNA binding, identifying regions that become protected or exposed during substrate recognition.

• Molecular dynamics simulations, as demonstrated in recent studies, can provide valuable insights into the flexibility and dynamics of the TSEN34-RNA interaction, complementing static structural data .

How can mutations in TSEN34 be designed to study structure-function relationships?

Strategic mutation design for TSEN34 should target key functional domains identified in recent structural studies:

• Catalytic site mutations: Targeting residues in the catalytic triad can create catalytically inactive variants useful for mechanistic studies. For example, mutations in the active site have been used to prevent cleavage while maintaining substrate binding in structural studies .

• RNA binding interface mutations: Altering residues that contact the pre-tRNA substrate can help delineate the contribution of specific interactions to substrate specificity and binding affinity. The extensive contacts between TSEN34 and the D-arm of tRNA provide numerous targets for such mutations .

• Subunit interface mutations: Modifying residues at the TSEN34-TSEN54 interface can probe the importance of this interaction for complex stability and function. The buried surface area of approximately 2230 Ų between these subunits suggests multiple candidate residues for mutation .

• Domain deletion studies: Truncation of the disordered region (residues 121-179) has been shown not to impair complex formation or activity in vitro, suggesting functional redundancy or accessory roles for this region . Similar targeted deletions can provide insights into domain-specific functions.

• Mutation of the long α helix (34H) near the tRNA elbow can help determine its role in substrate recruitment or sensing correct tRNA folding.

How does TSEN34 contribute to non-canonical RNA processing beyond tRNA splicing?

Recent research has uncovered roles for TSEN beyond canonical tRNA splicing. A bioinformatic analysis of RNA ends in yeast identified novel messenger RNA (mRNA) substrates of TSEN, with a conserved adenosine at the -1 position of the 3' cleavage site . Interestingly, these mRNA substrates were dependent on the catalytic activity of TSEN2 but not TSEN34, suggesting differential contributions of TSEN subunits to non-canonical substrate processing. The high structural plasticity of TSEN at the 5' splice site likely allows the enzyme complex to recognize substrates other than pre-tRNAs.
In yeast, TSEN has been implicated in the degradation of a subset of mRNAs encoding mitochondrial proteins, indicating a potential role in regulating mitochondrial function . Additionally, TSEN has an unknown essential function that appears to be linked to the integrated stress response and intron debranching, as revealed through genetic studies. This suggests that TSEN34, as part of the TSEN complex, may participate in stress response pathways through RNA processing mechanisms that are still being elucidated.

What is the relationship between TSEN34 mutations and pontocerebellar hypoplasia?

Mutations in all four TSEN subunits, including TSEN34, have been linked to pontocerebellar hypoplasia (PCH), a group of neurodegenerative disorders characterized by atrophy of the cerebellum and pons, microcephaly, developmental disorders, respiratory failure, and often early childhood death . While the exact mechanisms linking TSEN34 mutations to PCH remain incompletely understood, several hypotheses have emerged from research:

• Disruption of canonical tRNA processing may lead to translational defects that particularly affect rapidly developing neurons.

• Impaired processing of non-canonical RNA substrates may disrupt gene expression programs critical for neuronal development.

• Given the link between TSEN and the integrated stress response, mutations may compromise cellular adaptation to stress, particularly in neurons with high metabolic demands.
  Research using patient-derived mutations in experimental models can help elucidate the pathophysiological mechanisms. Understanding how specific TSEN34 mutations affect complex formation, catalytic activity, and substrate specificity may provide insights into the selective neuronal vulnerability observed in PCH patients.

How can cryo-EM structures inform the design of TSEN34 variants with altered specificity?

Recent cryo-EM structures of the TSEN complex bound to pre-tRNAs provide valuable blueprints for engineering TSEN34 variants with altered substrate specificity. The structures reveal that TSEN34 makes extensive contacts with the D-arm of tRNA and is positioned surrounding the 3' splice site . Based on these insights, researchers can:

What are the implications of TSEN34's dynamic structural features for its function?

The structural dynamics of TSEN34 appear crucial for its function in several ways:

• The large insertion between the N and C subdomains, which includes a long α helix (34H) positioned near the elbow of bound tRNA, likely facilitates substrate recruitment or sensing of correct tRNA tertiary structure . This dynamic element may function as a quality control mechanism, ensuring only properly folded tRNAs are processed.

• The lack of well-ordered density for the intron surrounding the 5' splice site in cryo-EM reconstructions suggests inherent flexibility in this region. This dynamic nature may be important for accommodating different substrates with varying intron sequences and lengths .

• Molecular dynamics simulations have shown that while the core structure of the TSEN complex remains rigid, there is considerable flexibility at the periphery, including the central domain of TSEN2 . This combination of rigid catalytic cores with flexible peripheral domains likely enables precise catalysis while maintaining adaptability in substrate recognition.

• The disordered regions in TSEN34 that are not visible in structural studies may serve as molecular recognition features for interactions with additional protein partners or regulatory factors, potentially coupling tRNA processing to other cellular pathways.

What are common challenges in expressing and purifying functional recombinant TSEN34, and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant TSEN34:

• Solubility issues: TSEN34 may form inclusion bodies during expression. This can be addressed by:

  • Lowering expression temperature (16-18°C)

  • Using solubility-enhancing fusion tags (such as SUMO or MBP)

  • Optimizing induction conditions (lower IPTG concentration, 0.1-0.5 mM)

  • Using specialized E. coli strains designed for membrane or difficult proteins

• Proteolytic degradation: The large insertion and disordered regions in TSEN34 may be susceptible to proteolysis. Mitigation strategies include:

  • Including protease inhibitors throughout purification

  • Reducing purification time and maintaining cold temperatures

  • Engineering constructs that remove highly susceptible regions while maintaining activity

• Lack of catalytic activity: When expressed alone, TSEN34 may lack activity due to absence of other TSEN subunits. Solutions include:

  • Co-expressing with other TSEN subunits

  • Reconstituting the complex in vitro with separately purified subunits

  • Using cell-free expression systems that allow simultaneous production of all subunits

• Aggregation during storage: Similar to other recombinant proteins, TSEN34 may aggregate during storage. To minimize this:

  • Store at high concentration (>1 mg/mL) with glycerol (10-20%)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Optimize buffer conditions through thermal stability assays

How can researchers distinguish between TSEN34-specific effects and those dependent on the intact TSEN complex?

Differentiating TSEN34-specific effects from those requiring the complete TSEN complex requires several complementary approaches:

• Comparative activity assays: Testing catalytic activity of TSEN34 alone versus the reconstituted complex on various substrates can reveal TSEN34-specific functions. While TSEN34 catalyzes 3' splice site cleavage, recent research shows that some mRNA substrates depend only on TSEN2 activity, not TSEN34 .

• Domain-specific mutations: Introducing mutations in TSEN34 that affect either its catalytic site or interfaces with other subunits can help dissect its independent functions versus collaborative roles.

• Subunit depletion studies: In cellular models, selective depletion of TSEN34 while maintaining other subunits (or vice versa) can help distinguish between subunit-specific and complex-dependent phenotypes.

• Cross-species complementation: Testing whether TSEN34 from one species can functionally substitute for its ortholog in another species, either alone or in the context of the complete complex, can provide insights into conserved independent functions.

• Binding partner identification: Immunoprecipitation of tagged TSEN34 under conditions that maintain or disrupt the TSEN complex can identify TSEN34-specific interacting partners versus complex-dependent interactions.

What experimental controls are essential when studying TSEN34 catalytic activity?

When assessing TSEN34 catalytic activity, several controls are critical for robust experimental design:

• Catalytically inactive mutant: A TSEN34 variant with mutations in key catalytic residues serves as a negative control to confirm that observed activity is due to TSEN34's enzymatic function rather than contaminants.

• Substrate specificity controls: Including both canonical (pre-tRNAs) and non-canonical RNA substrates helps define the specificity profile of TSEN34.

• Metal ion dependency: Since many nucleases require divalent metal ions, testing activity in the presence of metal chelators (such as EDTA) and with various metal ions helps characterize the catalytic mechanism.

• Temperature and pH controls: Assessing activity across a range of conditions ensures that experiments are conducted under optimal conditions and provides insights into stability and environmental sensitivity.

• RNA integrity controls: Using RNase inhibitors and including untreated RNA samples helps ensure that observed cleavage patterns are due to TSEN34 activity rather than contaminating nucleases or spontaneous degradation.

• Complex integrity verification: When working with the reconstituted TSEN complex, size exclusion chromatography or native PAGE should be used to confirm proper complex formation before activity assays.

What approaches can address data inconsistencies in TSEN34 functional studies?

When faced with inconsistent results in TSEN34 research, several systematic approaches can help resolve discrepancies:

• Protein quality assessment: Batch-to-batch variations in recombinant protein quality can significantly impact results. Implementing rigorous quality control through methods like thermal shift assays, dynamic light scattering, and activity benchmarking can identify problematic preparations.

• Substrate preparation standardization: Variations in RNA substrate preparation, particularly with respect to proper folding and potential contamination, can lead to inconsistent results. Standardizing annealing protocols and implementing quality controls for RNA substrates is essential.

• Complex stoichiometry verification: The functional TSEN complex requires specific stoichiometry of its subunits. Analytical techniques such as SEC-MALS (size exclusion chromatography with multi-angle light scattering) can verify proper complex formation with the correct subunit ratio.

• Experimental condition optimization: Systematic variation of buffer components, salt concentration, pH, and temperature can identify optimal and robust conditions for reproducible activity.

• Cross-validation with multiple assays: Using orthogonal assays to measure the same activity or interaction provides stronger evidence and can help identify assay-specific artifacts. For instance, complementing gel-based cleavage assays with fluorescence-based real-time kinetic measurements.

What are the most promising approaches for identifying novel TSEN34 substrates beyond tRNAs?

Several cutting-edge approaches show promise for discovering non-canonical TSEN34 substrates:

• CLIP-seq (UV crosslinking and immunoprecipitation followed by high-throughput sequencing): This approach can identify RNAs directly bound by TSEN34 in vivo, potentially revealing unexpected substrate classes. Recent bioinformatic analyses have already identified novel mRNA substrates of TSEN in yeast .

• RNA decay profiling: Comparing RNA populations in wild-type cells versus those with TSEN34 mutations can identify transcripts whose stability depends on TSEN34 activity. This approach has revealed that TSEN is involved in degrading a subset of mRNAs encoding mitochondrial proteins in yeast .

• In vitro selection approaches: Techniques like SELEX (Systematic Evolution of Ligands by Exponential Enrichment) using recombinant TSEN34 or the complete TSEN complex can identify RNA sequences or structures preferentially bound or cleaved.

• Structural biology-guided substrate prediction: Insights from cryo-EM structures of the TSEN-pre-tRNA complex can inform computational approaches to predict RNAs with structural features conducive to TSEN34 recognition .

• Genetic interaction screens: Synthetic genetic array (SGA) analysis or similar approaches can identify genes whose deletion exacerbates or suppresses phenotypes of TSEN34 mutations, potentially highlighting pathways involving non-canonical TSEN34 substrates.

How might the study of TSEN34 inform therapeutic approaches for pontocerebellar hypoplasia?

Understanding TSEN34's function and disease-associated mutations provides several avenues for therapeutic development:

• Small molecule modulators: Structural insights from cryo-EM studies could guide the design of compounds that stabilize mutant TSEN complexes or enhance their residual activity. The extensive interface between TSEN34 and TSEN54 (contact surface area of ~2287.2 Ų) offers potential binding sites for stabilizing compounds .

• RNA-based therapies: Antisense oligonucleotides or similar approaches might be developed to promote correct splicing of tRNAs or other substrates in cells with TSEN mutations, bypassing the compromised enzymatic activity.

• Gene therapy strategies: Delivery of functional TSEN34 to affected tissues, particularly in the developing brain, could potentially rescue the neurological phenotypes associated with TSEN34 mutations.

• Metabolic interventions: Given the link between TSEN and the integrated stress response , therapies targeting cellular stress pathways might mitigate the consequences of TSEN dysfunction, even without directly restoring TSEN activity.

• Compensatory approaches: Understanding the downstream effects of TSEN34 dysfunction might reveal compensatory pathways that could be therapeutically enhanced to bypass the need for TSEN34 activity.

What insights might TSEN34 provide into the evolution of RNA processing mechanisms?

TSEN34 offers a valuable window into evolutionary aspects of RNA processing:

• Ancient origins of splicing mechanisms: The conservation of TSEN homologs across eukaryotes and some archaea suggests that the basic mechanism of tRNA intron removal predates the divergence of these domains of life . Comparative studies of TSEN34 orthologs could illuminate how this ancient mechanism has been preserved and modified through evolution.

• Acquisition of new functions: The involvement of TSEN in processing non-tRNA substrates suggests functional diversification during evolution. Mapping these non-canonical functions across species could reveal when and how TSEN34 acquired new roles beyond tRNA splicing.

• Co-evolution with RNA substrates: Analyzing how TSEN34 structure relates to the distribution and features of intron-containing tRNAs across species could provide insights into the co-evolution of the enzyme and its substrates.

• Integration with cellular stress responses: The link between TSEN and the integrated stress response suggests that RNA processing mechanisms have been incorporated into cellular homeostatic networks during evolution. Understanding this integration could reveal general principles about how ancient enzymatic activities become incorporated into newer regulatory pathways.

• Structural adaptations: The cryo-EM structures of human TSEN reveal specific adaptations compared to archaeal orthologs, such as the extensive interface between TSEN34 and TSEN54 . Analyzing these adaptations across evolutionary time can provide insights into how protein complexes evolve to accommodate new functions while maintaining core activities.