Recombinant Debaryomyces hansenii tRNA guanosine-2'-O-methyltransferase TRM13 (TRM13)

Basic Research Questions

• What is D. hansenii TRM13 and what is its primary function?

  D. hansenii TRM13 is a tRNA methyltransferase that catalyzes the 2'-O-methylation of specific nucleotides at position 4 in tRNAs. Based on homology with the Saccharomyces cerevisiae ortholog, it belongs to the Rossmann-fold methyltransferase (RFM) superfamily . This enzyme plays a crucial role in post-transcriptional modification of tRNAs, which affects their stability, folding, and function in translation. D. hansenii, being a halotolerant yeast commonly found in cheese and marine environments, likely possesses a TRM13 variant adapted to function efficiently under high salt conditions .

• How does D. hansenii TRM13 compare to homologous proteins in other yeasts?

  While the core catalytic function is conserved among TRM13 homologs across different yeast species, D. hansenii TRM13 likely possesses unique characteristics related to the halotolerant nature of this organism. From bioinformatics analysis of the Saccharomyces ortholog, we know that Trm13 is a strongly diverged member of the RFM superfamily, with conserved residues in the predicted active site suggesting it may use a different mechanism of ribose methylation than its relatives . Given D. hansenii's adaptation to high-salt environments (tolerating up to 4.11 M sodium), its TRM13 might have evolved specific structural features that allow optimal function under such conditions .

• What substrates does D. hansenii TRM13 typically modify?

  Based on homology to Trm13 in other yeasts, D. hansenii TRM13 likely modifies specific tRNAs at position 4. In human cells, Trmt13 primarily modifies cytoplasmic tRNA^His, tRNA^Pro, and tRNA^Gly, with evidence also showing modification of tRNA^Arg, tRNA^Lys, and tRNA^Thr . The specific pattern of substrate recognition involves the CHHC zinc finger domain, which is crucial for tRNA binding . Experimental verification of D. hansenii TRM13's exact substrate profile would require methods such as RNA-mass spectrometry after enzyme incubation with various tRNA substrates, following approaches similar to those used for human Trmt13 characterization .

• What genetic tools are available for studying TRM13 in D. hansenii?

  Recent advances have significantly improved the genetic toolkit for D. hansenii, facilitating TRM13 studies:

  Genetic Tool	Description	Reference
  CRISPR/Cas9 system	Plasmid-based CRISPR^CUG/Cas9 method for efficient gene editing
  KU70 knockout strain	NHEJ-deficient strain improving homologous recombination efficiency
  Transformation system	Based on histidine auxotrophic recipient strain and DhHIS4 marker
  Expression vectors	TEF1 promoter (A. adeninivorans) with CYC1 terminator for optimal expression
  In vivo DNA assembly	Co-transformation of DNA fragments with 30-bp homologous overlaps

  These tools allow for gene deletion, point mutation introduction, and protein tagging approaches that can be applied to TRM13 functional studies .

• How is TRM13 expression regulated in D. hansenii under different salt conditions?

  D. hansenii exhibits significant transcriptomic and proteomic changes in response to salt stress, which likely affects TRM13 expression. In chemostat cultivations with 1M NaCl or KCl, D. hansenii shows distinct expression profiles, with sodium and potassium triggering different responses at both gene expression and protein activity levels . While specific data on TRM13 regulation is not directly reported in the literature, the cellular response suggests an adapted gene expression network that supports growth under high salt conditions. Interestingly, D. hansenii shows improved performance against various stresses (extreme pH, oxidative stress, temperature) when in the presence of 1M NaCl, indicating salt-induced protective mechanisms that may involve RNA modification enzymes like TRM13 .

Advanced Research Questions

• What methods are most effective for expressing recombinant D. hansenii TRM13?

  For optimal expression of recombinant D. hansenii TRM13, consider the following methodological approach:

  1. Expression System Selection:

     • For homologous expression, use D. hansenii itself, leveraging its native machinery

     • For heterologous expression, E. coli systems may require codon optimization due to the alternative genetic code usage in D. hansenii (CUG clade)

  2. Vector Construction:

     • Utilize the plasmid-based CRISPR^CUG/Cas9 method developed for D. hansenii

     • Include the TEF1 promoter from Arxula adeninivorans for highest production yield

     • Consider the panARS element for plasmid maintenance

  3. Expression Conditions:

     • Culture at pH 4 with 1M NaCl for optimal growth conditions

     • Growth temperature between 20-30°C, with 25°C being optimal for most strains

     • Consider the strain-specific responses to sodium; some strains (e.g., IBT27) show significantly enhanced growth in acidic pH with high sodium content compared to standard strain CBS767

  4. Purification Strategy:

     • Include salt (0.5-1M NaCl) in extraction and purification buffers to maintain protein stability

     • Use affinity chromatography (His-tag) followed by size exclusion for highest purity

• How can CRISPR/Cas9 be used to study TRM13 function in D. hansenii?

  CRISPR/Cas9 technology provides powerful approaches for studying TRM13 function in D. hansenii:

  1. System Selection:

     • Use the plasmid-based CRISPR^CUG/Cas9 method specifically developed for D. hansenii

     • This system uses a dominant NAT marker allowing selection in prototrophic strains

  2. sgRNA Design:

     • Target the TRM13 gene with multiple sgRNAs to increase editing efficiency

     • The system supports expressing multiple sgRNAs from a single vector for multiplex gene editing

  3. Editing Approaches:

     • For gene deletion: Target coding regions with two sgRNAs and provide a repair template

     • For point mutations: 90-nt single-stranded oligonucleotides are sufficient for precise edits with up to 100% efficiency

     • For functional domain analysis: Create targeted mutations in the methyltransferase domain or zinc finger domains based on homology to characterized Trm13 proteins

  4. Enhanced Editing Efficiency:

     • Use a KU70-deficient strain to significantly improve homologous recombination efficiency

     • This approach enables marker-free gene deletions and precise point mutations

  5. Functional Analysis:

     • Assess tRNA methylation using RNA-mass spectrometry or primer extension assays

     • Compare growth rates and stress responses between wild-type and TRM13 mutant strains

     • Analyze effects on protein synthesis using polysome profiling or puromycin incorporation assays

• What experimental approaches can be used to characterize the catalytic activity of D. hansenii TRM13?

  Comprehensive characterization of D. hansenii TRM13 catalytic activity requires multiple complementary approaches:

  1. In vitro Methyltransferase Assays:

     • Radioactive assay using [³H]- or [¹⁴C]-SAM as methyl donor

     • Non-radioactive alternative using SAM analogs with detectable moieties

     • RNA-mass spectrometry to detect specific modifications in tRNAs post-reaction

     • HPLC-based detection of modified nucleosides after enzymatic digestion of tRNAs

  2. Substrate Specificity Analysis:

     • Test modification of various tRNAs (tRNA^His, tRNA^Pro, tRNA^Gly, tRNA^Arg, tRNA^Lys, tRNA^Thr)

     • Use site-directed mutagenesis of target nucleotides to confirm position specificity

     • Electrophoretic mobility shift assays (EMSA) to assess binding affinity to different tRNAs

  3. Domain Function Analysis:

     • Create deletion or point mutations in zinc finger domains and assess effects on activity

     • Based on human Trmt13 studies, the CHHC zinc finger domain is essential for tRNA binding

     • The E463A mutation in the catalytic domain eliminates methyltransferase activity

  4. Condition Dependency:

     • Test activity across a range of salt concentrations (0-2M NaCl)

     • Compare activity with different salt types (NaCl vs KCl)

     • Analyze pH (4.5-6.0) and temperature (20-35°C) effects on enzyme activity

• How does salt concentration affect the activity and stability of D. hansenii TRM13?

  D. hansenii's halotolerant nature suggests its TRM13 enzyme has adapted to function under high salt conditions:

  1. Activity Analysis:

     • Systematically test TRM13 methyltransferase activity across 0-2M NaCl concentrations

     • Compare NaCl vs KCl effects, as D. hansenii exhibits different responses to these salts

     • Determine optimal salt concentration for maximum catalytic efficiency

  2. Structural Stability:

     • Assess thermal stability using differential scanning fluorimetry at various salt concentrations

     • Monitor protein folding using circular dichroism spectroscopy under different salt conditions

     • Evaluate aggregation propensity using dynamic light scattering techniques

  3. Kinetic Parameters:

     • Determine Km and Vmax values for tRNA substrates under varying salt concentrations

     • Analyze the effect of salt on SAM binding efficiency

     • Investigate whether salt affects substrate specificity or merely catalytic rate

  4. Molecular Adaptation Analysis:

     • Compare with TRM13 homologs from non-halotolerant yeasts under identical conditions

     • Identify specific amino acid residues that contribute to salt tolerance

     • This approach aligns with observations that D. hansenii shows improved performance under various stresses in the presence of 1M NaCl

• What are the challenges in creating TRM13 knockout strains in D. hansenii?

  Creating TRM13 knockout strains in D. hansenii presents several challenges that researchers need to address:

  1. DNA Repair Mechanism Bias:

     • D. hansenii preferentially uses non-homologous end-joining (NHEJ) DNA repair

     • This poses challenges for precise gene targeting via homologous recombination

     • The development of NHEJ-deficient strains (KU70 knockout) has significantly improved targeting efficiency

  2. Transformation Efficiency:

     • Historically, D. hansenii has been difficult to transform efficiently

     • Recent methods include electroporation-based approaches with reported success

     • Selection of appropriate vectors with suitable origins of replication is critical

  3. Selection System:

     • Limited availability of selection markers appropriate for D. hansenii

     • Recent developments include:

       • Histidine auxotrophic strain (DBH9) with DhHIS4 as selectable marker

       • NAT resistance marker compatible with prototrophic strains

       • Hygromycin resistance for selection in D. hansenii

  4. Validation Approaches:

     • PCR verification of gene deletion

     • Functional validation through RNA methylation analysis

     • Phenotypic characterization under various growth conditions

     • RNA-MS to confirm absence of TRM13-specific methylation patterns

• How can the dual roles of TRM13 in transcription and translation be studied in D. hansenii?

  Based on studies of human Trmt13 showing dual roles in transcription and translation , investigating similar functions in D. hansenii TRM13 requires:

  1. Subcellular Localization:

     • Create fluorescently tagged TRM13 to visualize localization

     • Perform subcellular fractionation followed by Western blotting

     • Analyze whether salt stress affects subcellular distribution

  2. RNA Modification Analysis:

     • Use RNA-mass spectrometry to identify and quantify TRM13-dependent modifications

     • Perform primer extension assays under low dNTP conditions to detect 2'-O-methylation sites

     • Compare modification patterns between wild-type and TRM13 mutant strains

  3. Chromatin Association:

     • Perform chromatin immunoprecipitation (ChIP) to identify potential DNA binding sites

     • In human cells, Trmt13 functions as a transcriptional co-activator by binding DNA through its CHHC zinc finger domain

     • ChIP-seq analysis to identify genome-wide binding patterns

  4. Domain-Specific Mutations:

     • Create the catalytically inactive E463A mutation (based on human ortholog) that eliminates methyltransferase activity but may preserve DNA binding

     • Delete or mutate the CHHC zinc finger domain to disrupt DNA/RNA binding

     • Generate domain swap chimeras to identify regions responsible for specific functions

  5. Translation Analysis:

     • Assess global protein synthesis using puromycin incorporation assays

     • Analyze polysome profiles to evaluate translation efficiency

     • Investigate the formation and levels of specific tRNA fragments (tRFs) that may regulate translation

• How does D. hansenii TRM13 activity correlate with stress response pathways?

  D. hansenii's remarkable tolerance to multiple stresses suggests TRM13 may play a role in stress response mechanisms:

  1. Stress Response Integration:

     • Analyze TRM13 expression and activity under various stress conditions (salt, oxidative, pH, temperature)

     • D. hansenii shows improved performance against various stresses when in the presence of 1M NaCl

     • Investigate whether TRM13-mediated tRNA modifications change under stress conditions

  2. Comparative Stress Analysis:

     • Compare wild-type and TRM13 mutant strains under various stress conditions

     • Measure growth rates, viability, and metabolism

     • The strain-specific responses to sodium observed in D. hansenii (e.g., IBT27 vs CBS767) may correlate with differences in TRM13 activity

  3. Signaling Pathway Interactions:

     • Investigate potential phosphorylation of TRM13 under stress conditions

     • Human Trmt13 shows regulated activity via post-translational modifications

     • Use phosphoproteomic approaches similar to those employed in D. hansenii salt response studies

  4. tRNA Fragment Analysis:

     • Quantify stress-induced tRNA fragments (tRFs) in wild-type vs TRM13 mutants

     • In human cells, Trmt13 regulates the level of specific tRFs that affect protein synthesis

     • Use modified RT-qPCR with stem-loop primers to accurately detect tRF levels

• What bioinformatic approaches can identify functional motifs in D. hansenii TRM13?

  Computational analysis of D. hansenii TRM13 can provide valuable insights into its function:

  1. Sequence Analysis:

     • Multiple sequence alignment of TRM13 homologs across species

     • Identify conserved domains, particularly the methyltransferase domain and zinc finger motifs

     • Based on Saccharomyces Trm13p analysis, expect a Rossmann-fold methyltransferase (RFM) structure

  2. Structural Modeling:

     • Generate homology models based on related methyltransferases

     • For Saccharomyces Trm13p, a molecular model was constructed to facilitate experimental analyses

     • Use model quality assessment methods to evaluate structural predictions

  3. Functional Motif Prediction:

     • Identify SAM-binding motifs characteristic of methyltransferases

     • Analyze zinc finger domains (CCCH, CHHC) for RNA/DNA binding potential

     • Predict potential interaction sites with tRNA substrates

  4. Comparative Genomics:

     • Analyze TRM13 conservation across yeast species with varying salt tolerance

     • Identify D. hansenii-specific sequence features that might correlate with halotolerance

     • Compare with genomic data from the DebaryOmics study to identify potential co-regulated genes

  5. Post-translational Modification Prediction:

     • Identify potential phosphorylation sites that might regulate TRM13 activity

     • Recent phosphoproteomic analysis in D. hansenii has implicated novel mechanisms in salt response

     • Predict subcellular localization signals that might direct dual functionality