Recombinant Xenopus laevis Threonylcarbamoyladenosine tRNA methylthiotransferase (cdkal1)

What is CDKAL1 and what is its function in Xenopus laevis?

CDKAL1 in Xenopus laevis is a methylthiotransferase enzyme belonging to the radical S-adenosylmethionine (SAM) family. Its primary function is catalyzing the addition of a methylthio (-SCH3) group to threonylcarbamoyladenosine (t6A) residues at position 37 of specific tRNAs, producing 2-methylthio-N-threonylcarbamoyladenosine (ms2t6A) . This modification is particularly important in tRNAs that decode ANN codons. In the broader context of methylthiotransferases, these enzymes are phylogenetically classified into several clades, including RimO, MiaB, MtaB, and e-MtaB, with CDKAL1 belonging to the e-MtaB clade found in eukaryotes and archaea . This post-transcriptional modification enhances translational fidelity and efficiency by stabilizing codon-anticodon interactions during protein synthesis.

What expression systems are recommended for producing recombinant Xenopus laevis CDKAL1?

For recombinant expression of Xenopus laevis CDKAL1, several expression systems can be employed based on experimental requirements:

• E. coli expression system: This is commonly used due to its simplicity and high yield. For example, expression can be achieved using E. coli-CodonPlus (DE3)-RIL strains with pET15b vectors carrying an N-terminal hexahistidine tag, as demonstrated with other MTTases . Culture conditions typically involve growth in LB medium supplemented with appropriate antibiotics (e.g., 100 μg/mL ampicillin) at 37°C until reaching an OD600 of ~0.4, followed by addition of cysteine and FeCl3 (25 μM each) and IPTG induction (100 μM) at an OD600 of ~0.7 .

• Xenopus expression system: For more native-like post-translational modifications, expression in Xenopus cells or embryos can be achieved using transgenic approaches. Methods include restriction enzyme mediated integration (REMI), phiC31 integrase, I-SceI meganuclease, or transposable element-based approaches . I-SceI meganuclease has shown high efficiency with integration rates of up to 20% in X. laevis .

• Alternative archaeal systems: For functional studies comparing CDKAL1 with archaeal homologs, expression in systems like Methanococcus maripaludis (as used for MjMTTase) can provide valuable comparative data .

What purification methods are most effective for isolating recombinant CDKAL1?

Purification of recombinant CDKAL1 requires careful consideration of its oxygen sensitivity and iron-sulfur cluster content. Based on established protocols for similar MTTases, the following methods are recommended:

• Anaerobic purification: Conduct all purification steps inside an anaerobic chamber with an atmosphere of N2 or similar inert gas to preserve the Fe-S clusters .

• Affinity chromatography options:

  • His-tag purification: For His-tagged constructs, use Ni-NTA resin with anaerobic buffers containing reducing agents like DTT or 2-mercaptoethanol.

  • Strep-tag purification: For Strep-tagged constructs, Strep-Tactin Sepharose resin can be used with Strep buffer W (100 mM Tris, 150 mM NaCl, pH 8.0) for washing and elution with 2.5 mM desthiobiotin .

• Post-purification processing: Concentrate purified protein using appropriate molecular weight cutoff filters (e.g., 30 kD Amicon Ultra) and exchange into storage buffer (e.g., 50 mM HEPES, 300 mM KCl, 5 mM DTT, pH 8.0) using desalting columns .

• Storage: Store purified protein in anaerobic cryovials, flash freeze in liquid N2, and maintain at -80°C to preserve activity .

How can the enzymatic activity of purified CDKAL1 be measured in vitro?

The enzymatic activity of purified CDKAL1 can be assessed through several complementary approaches:

• Substrate preparation: Prepare substrates by in vitro transcription of tRNAs followed by enzymatic incorporation of t6A modification using the KEOPS/EKC complex or TsaD enzyme .

• In vitro activity assay: Set up reactions containing:

  • Purified CDKAL1 (1-5 μM)

  • Modified tRNA substrate (5-10 μM)

  • S-adenosylmethionine (SAM) (1-2 mM)

  • Sodium dithionite (1-5 mM) as electron donor

  • DTT (5 mM)

  • Buffer (e.g., 50 mM HEPES, 100 mM KCl, pH 7.5)

• Analysis methods:

  • HPLC-MS/MS: Digest tRNA with nuclease P1 and analyze nucleoside composition to detect ms2t6A formation

  • Radiometric assays: Using [methyl-14C]-SAM to track methyl transfer

  • Gel shift assays: To observe migration differences between unmodified and modified tRNAs

• Controls: Include negative controls (reactions without enzyme or SAM) and positive controls (known active MTTase with its substrate) to validate assay performance.

How do the substrate specificity determinants of Xenopus CDKAL1 compare to MTTases from other domains of life?

The substrate specificity of Xenopus CDKAL1 compared to MTTases from other domains of life reveals important evolutionary and functional insights:

• Substrate recognition patterns:

  • CDKAL1 (e-MtaB clade): Primarily recognizes t6A-containing tRNAs that decode ANN codons . The enzyme likely recognizes both the t6A modification and specific structural elements of the tRNA anticodon stem-loop.

  • Bacterial MtaB: Similar to e-MtaB but with distinct sequence preferences. Some bacterial MtaB enzymes can also recognize and modify hn6A-containing tRNAs to produce ms2hn6A .

  • Bacterial MiaB: Recognizes i6A-containing tRNAs that decode UNN codons to produce ms2i6A, showing distinct substrate preference from CDKAL1 .

  • RimO: Uniquely targets protein substrates (D88 of ribosomal protein S12) rather than tRNA .

• Comparative activity table:

• Structural basis for specificity:
  Analysis of protein sequences and structural models suggests that substrate specificity is determined by specialized domains outside the core radical SAM domain. The UPF0004 domain likely plays a key role in tRNA recognition, while differences in the C-terminal TRAM domain contribute to distinguishing between different modified adenosine substrates .

What are the most effective gene editing approaches for studying CDKAL1 function in Xenopus laevis?

Given the allotetraploid nature of Xenopus laevis and the importance of CDKAL1 in tRNA modification, the following gene editing approaches are most effective for functional studies:

• CRISPR/Cas9-based approaches:

  • Dual targeting strategy: Design sgRNAs targeting conserved regions in both homeologs (L and S chromosomes) of X. laevis CDKAL1 .

  • Delivery method: Microinjection of Cas9 protein and sgRNA complexes into fertilized eggs at the one-cell stage.

  • Screening approach: T7 endonuclease assay or direct sequencing to identify mutations.

• Gene replacement strategies:

  • While random integration has been the standard for Xenopus transgenesis, targeted approaches using homology-directed repair with CRISPR/Cas9 have recently been described, allowing precise genetic modifications .

  • For complementation studies, I-SceI meganuclease-mediated transgenesis has shown integration rates of up to 20% in X. laevis and is effective for rescue experiments .

• Conditional knockout approaches:

  • For studying developmental roles without lethal phenotypes, consider tissue-specific or inducible knockout strategies using Cre-loxP or similar systems.

  • Tissue-specific promoters available for Xenopus include those from transgenic lines like Xla.Tg(Dre.cdh17:eGFP) for kidney-specific expression .

• Experimental validation of editing:

  • Molecular verification: RT-PCR, Western blotting

  • Functional verification: Analysis of tRNA modifications by HPLC-MS/MS to confirm reduction in ms2t6A levels

  • Phenotypic analysis: Developmental timing, morphology, and tissue-specific defects

What is the relationship between CDKAL1 activity and the Fe-S cluster assembly machinery in Xenopus systems?

The activity of CDKAL1, like other radical SAM enzymes, critically depends on iron-sulfur (Fe-S) clusters. In Xenopus systems, this relationship involves several key aspects:

• Fe-S cluster requirements:

  • CDKAL1 contains at least two [4Fe-4S] clusters: one for SAM binding and radical generation, and a second auxiliary cluster likely involved in sulfur mobilization for the methylthio group .

  • The clusters are oxygen-sensitive, necessitating anaerobic conditions during purification and enzymatic assays .

• Fe-S cluster assembly systems in Xenopus:

  • Xenopus employs multiple Fe-S cluster assembly systems:

    • Mitochondrial ISC (Iron-Sulfur Cluster) machinery

    • Cytosolic CIA (Cytosolic Iron-sulfur protein Assembly) pathway

    • Potential involvement of the SUF (Sulfur mobilization) pathway components

• Experimental approaches to study this relationship:

  • Genetic knockdown: Target components of Fe-S assembly machinery (e.g., ISCU, NFS1, NARFL) to observe effects on CDKAL1 activity.

  • Chemical inhibition: Use compounds that disrupt Fe-S cluster formation (e.g., deferoxamine) to assess impact on CDKAL1.

  • Fe-S transfer studies: Investigate whether dedicated carrier proteins deliver clusters to CDKAL1.

  • Spectroscopic analysis: Use UV-visible spectroscopy, EPR, and Mössbauer spectroscopy to characterize CDKAL1's Fe-S clusters under various conditions.

• Correlation with developmental expression:

  • Study the co-expression patterns of CDKAL1 and Fe-S assembly machinery components during Xenopus development to identify critical periods.

How does the methylthiolation activity of CDKAL1 impact tRNA function and translational fidelity in Xenopus developmental contexts?

The methylthiolation of t6A to ms2t6A by CDKAL1 has significant implications for tRNA function and translational fidelity during Xenopus development:

• Molecular impact on tRNA structure and function:

  • The ms2t6A modification enhances base stacking and stabilizes anticodon-codon interactions, particularly for ANN codons .

  • This modification can influence tRNA recognition by the ribosome and potentially alter translation elongation rates at specific codons.

• Developmental stage-specific requirements:

  • Temporal expression analysis: Study CDKAL1 expression and ms2t6A modification levels across developmental stages from early cleavage through organogenesis.

  • Tissue-specific patterns: Examine whether certain tissues (e.g., pancreas, neural tissue) have higher requirements for CDKAL1 activity, similar to tissue-specific effects observed in mammalian systems.

• Experimental approaches to assess translational impact:

  • Ribosome profiling: Compare ribosome distribution on mRNAs between wild-type and CDKAL1-deficient embryos to identify codons with altered translation efficiency.

  • Proteomics: Quantitative mass spectrometry to detect changes in protein expression or modification patterns.

  • Reporter assays: Construct reporters with codon bias towards ANN codons to detect changes in translational efficiency or accuracy.

• Developmental phenotypes associated with CDKAL1 deficiency:

  • Early development: Potential cell division timing anomalies if specific maternal proteins are inefficiently translated.

  • Organogenesis: Based on mammalian studies, particular attention should be paid to pancreatic development, insulin signaling, and neural development.

  • Stress response: Investigate whether CDKAL1-deficient embryos show altered stress responses, as translational fidelity becomes increasingly important under stress conditions.

What methods can be used to analyze the structural basis of substrate recognition by Xenopus CDKAL1?

Understanding the structural basis of substrate recognition by Xenopus CDKAL1 requires multiple complementary approaches:

• Protein crystallography or cryo-EM:

  • Sample preparation: Crystallize purified CDKAL1 alone or in complex with tRNA substrate, SAM, and other cofactors under anaerobic conditions to preserve Fe-S clusters.

  • Structure determination: Use X-ray crystallography or cryo-electron microscopy to determine atomic-resolution structures.

  • Complex analysis: Particular focus on the interaction interfaces between CDKAL1 and the tRNA anticodon stem-loop region.

• Computational approaches:

  • Homology modeling: Build models based on related MTTase structures (e.g., MiaB, RimO) when direct structural data is unavailable.

  • Molecular dynamics simulations: Simulate protein-tRNA interactions to identify key binding determinants and conformational changes.

  • Sequence conservation analysis: Compare CDKAL1 sequences across species to identify evolutionarily conserved residues likely involved in substrate recognition.

• Biochemical and biophysical approaches:

  • Mutagenesis studies: Systematically mutate residues predicted to be involved in substrate recognition and test effects on binding and catalysis.

  • UV crosslinking: Map protein-RNA interaction sites using UV-induced crosslinking followed by mass spectrometry.

  • Binding assays: Use techniques like isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or microscale thermophoresis (MST) to measure binding affinities between CDKAL1 and various tRNA substrates.

• Substrate modification analysis:

  • Generate tRNA variants with modifications to the anticodon loop, T-loop, or other regions to identify critical recognition elements.

  • Test recognition of substrates with different modifications (t6A, hn6A, i6A) to define specificity determinants.