Recombinant Aspergillus niger Cytoplasmic tRNA 2-thiolation protein 1 (ncs6)

How is tRNA thiolation mediated by the Ncs2/Ncs6 complex?

The Ncs2/Ncs6 complex mediates tRNA thiolation through a conserved pathway that involves the Uba4-Urm1-Ncs2/Ncs6 components. In this process:

• The complex specifically modifies three tRNAs: tKUUU, tQUUG, and tEUUC

• The thiolation occurs at the wobble uridine position (U34)

• This modification enhances codon recognition and translation efficiency

Studies in M. oryzae demonstrate that deletion of either NCS2 or NCS6 leads to complete loss of tRNA thiolation, indicating both components are essential for this process . The Ncs2/Ncs6 complex likely forms a heterodimer, as confirmed by co-immunoprecipitation experiments in M. oryzae .

What methods are most effective for studying tRNA thiolation in A. niger?

For researchers investigating tRNA thiolation in A. niger, several methodological approaches have proven effective:

Detection of tRNA Thiolation:

• HPLC-coupled mass spectrometry of total tRNA can definitively identify thiolation modifications

• Northern blotting using denaturing PAGE gels supplemented with APM (N-acryloylamino phenyl mercuric chloride) can assess thiolation status in individual tRNAs, as thiolated tRNAs show reduced mobility due to affinity of the mercuric compound for the thio group

Genetic Manipulation:

• CRISPR/Cas9-based genome editing is effective for generating deletion mutants in A. niger, as demonstrated in studies of other genes in this organism

• PEG-mediated protoplast transformation can be used to introduce genetic modifications, though some A. niger strains (e.g., CBS112.48 and CBS769.97) may present difficulties in protoplasting

Expression Analysis:

• RT-qPCR can track dynamic expression patterns of NCS2 and NCS6 during different developmental stages

• RNA-seq and ribosome profiling can be combined to analyze how tRNA thiolation affects translation efficiency of genes containing AAA/CAA/GAA codons

What are the challenges in expressing recombinant Ncs6 from A. niger?

Expressing recombinant Ncs6 from A. niger presents several technical challenges:

• Structural Considerations: The Ncs6 protein likely has a complex structure similar to homologs in other fungi, which may affect proper folding when expressed recombinantly

• Functional Dependency: Since Ncs6 functions as part of a complex with Ncs2, expressing Ncs6 alone may not yield a functionally active protein

• Strain Selection: Careful selection of A. niger strains is crucial, as there is significant genetic diversity between strains (average of 6.1 ± 2.0 variants/kb) , which may affect Ncs6 sequence and function

• Protoplasting Difficulties: Some A. niger strains present challenges in protoplasting , which may complicate transformation procedures for expression systems

• Expression Verification: Since antibodies specific to A. niger Ncs6 may not be readily available, researchers often need to use epitope tags for detection and purification, which may affect protein function

How does deletion of ncs6 affect A. niger phenotype and metabolism?

Based on studies in related fungi like M. oryzae, deletion of ncs6 in A. niger would likely result in:

• Complete Loss of tRNA Thiolation: All three target tRNAs (tKUUU, tQUUG, tEUUC) would lack thiolation modifications

• Translation Efficiency Effects: Genes enriched in AAA/CAA/GAA codons would likely show reduced translation efficiency, as observed in M. oryzae

• Codon-Specific Ribosome Pausing: Increased ribosome pausing would be expected at CAA codons but not at synonymous CAG codons

• Developmental Impacts: Given that NCS6 shows dynamic expression during developmental stages in M. oryzae , deletion may affect specific developmental processes in A. niger

• Potential Impact on Sexual Development: Since A. niger has both mating types distributed across different phylogenetic clades , and given the role of proper gene expression in fungal development, NCS6 deletion might affect sexual reproduction processes if successfully induced

How does tRNA thiolation influence codon usage and translation efficiency in A. niger?

The influence of tRNA thiolation on codon usage and translation in A. niger likely follows patterns observed in other fungi:

Codon-Specific Effects:

• Genes with high frequency of AAA/CAA/GAA codons (decoded by thiolated tRNAs) would be most affected by loss of tRNA thiolation

• Reporter assays using constructs with consecutive CAA or CAG codons would show that CAA codons (decoded by thiolated tRNA) are more sensitive to loss of thiolation than CAG codons

Translation Efficiency:

• Without thiolation, genes containing high proportions of AAA/CAA/GAA codons would show decreased ribosome profiling (RPF) levels, indicating reduced translation efficiency

• These effects are codon-specific rather than gene-specific, as synonymous codons (AAG/CAG/GAG) would not show the same sensitivity to thiolation status

Gene Expression Classification:
The following table illustrates the expected relationship between codon usage and translation effects in thiolation-deficient mutants:

This pattern indicates that tRNA thiolation specifically promotes translation of genes enriched in AAA/CAA/GAA codons .

How conserved is the Ncs6 protein across different A. niger strains?

While specific data on Ncs6 conservation across A. niger strains is not directly available in the provided search results, we can make informed inferences:

What is the relationship between Ncs6 function and strain compatibility in A. niger?

The relationship between Ncs6 function and heterokaryon compatibility in A. niger presents an intriguing research avenue:

• A. niger exhibits widespread heterokaryon incompatibility, with only 1 out of 23 attempted parasexual crosses between different strains resulting in successful formation of heterokaryotic mycelium

• Since tRNA thiolation affects translation efficiency in a codon-specific manner , variations in Ncs6 function between strains could potentially impact heterokaryon compatibility by affecting the expression of genes involved in compatibility systems

• The single successful parasexual cross between A. niger CBS147323 and CBS147347 (both from clade B) could serve as a model system to investigate whether tRNA thiolation plays a role in compatible strain interactions

• Researchers could introduce NCS6 mutations or deletions in these compatible strains to determine if tRNA thiolation affects the success rate of heterokaryon formation or subsequent diploid stability

• This approach could provide insights into whether post-transcriptional modifications like tRNA thiolation contribute to reproductive barriers between fungal strains

How can CRISPR-Cas9 be used to study Ncs6 function in A. niger?

CRISPR-Cas9 technology offers powerful approaches for studying Ncs6 function in A. niger:

• Gene Deletion: Complete knockout of NCS6 can be achieved using CRISPR-Cas9, with transformants selected based on phenotypic changes resulting from indels generated by CRISPR-Cas9 endonuclease activity

• Domain Mutagenesis: Rather than deleting the entire gene, researchers can target specific functional domains within Ncs6 to determine their importance for tRNA thiolation

• Promoter Modification: CRISPR-Cas9 can be used to modify the NCS6 promoter to create conditional expression strains, enabling temporal control of Ncs6 function

• Fluorescent Tagging: C-terminal tagging of Ncs6 with fluorescent proteins can help visualize its subcellular localization, as demonstrated for other proteins in fungi

• Combined Marker Approach: As demonstrated in A. niger strain engineering, CRISPR-Cas9 can be combined with selection markers (like conidial color markers fwnA and brnA or auxotrophic markers pyrG and nicB) to facilitate identification of successfully edited strains

Practical Considerations:

• Protoplasting efficiency varies between A. niger strains, with some strains being particularly difficult to protoplast

• The presence of an intact kusA gene in wild-type strains allows for selection based on phenotype changes without providing repair DNA templates

• Multiple guide RNAs may be required for efficient editing, especially for genes in regions with low CRISPR efficiency

What experimental approaches can determine if tRNA thiolation affects sexual development in A. niger?

Investigating the potential role of tRNA thiolation in sexual development of A. niger requires specialized experimental approaches:

• Sclerotia Induction: Triton X-100 supplementation (0.05-1%) in media like MEA, PDA, or OA has been shown to induce sclerotia formation in A. niger , which is a prerequisite for sexual reproduction

• Diploid Development: Generate NCS6 deletion mutants in compatible A. niger strains (such as CBS147323 and CBS147347) with opposite mating types, then create heterozygous diploids through parasexual crossing

• Comparative Analysis: Compare sclerotia formation and potential ascospore development between:

  • Wild-type heterozygous diploids

  • Heterozygous diploids with one functional NCS6 copy

  • Diploids with both NCS6 copies deleted

• Media Optimization: Test various media combinations with Triton X-100 supplementation (MEA, PDA, OA, CYA, CYA/OA, WATM) to identify optimal conditions for sexual development

• Microscopic Analysis: Crack sclerotia on microscope slides with physiological salt buffer and use light microscopy to assess for presence of asci/ascospores

• Gene Expression Analysis: Compare expression profiles of genes involved in sexual development between wild-type and Ncs6-deficient strains during sclerotia formation using RNA-seq

• Translation Efficiency Impact: Use ribosome profiling to determine if genes essential for sexual development have high AAA/CAA/GAA codon usage that might make them particularly sensitive to tRNA thiolation status

How can researchers distinguish between direct and indirect effects of Ncs6 deletion?

Distinguishing between direct and indirect effects of Ncs6 deletion presents a significant analytical challenge:

• Codon Enrichment Analysis: Calculate the frequency of AAA/CAA/GAA codons in differentially expressed genes. Those with high frequency are likely direct targets of tRNA thiolation deficiency

• Ribosome Profiling and RNA-seq Integration: Combine ribosome profiling (RPF) with RNA-seq to classify affected genes:

  • Direct effects: Changes in RPF levels without corresponding changes in mRNA levels

  • Indirect effects: Concordant changes in both RPF and mRNA levels

• Reporter Assays: Use reporter constructs with varying frequencies of AAA/CAA/GAA codons to quantify translation effects directly attributable to thiolation status

• Temporal Analysis: Monitor gene expression and phenotypic changes at different time points after Ncs6 deletion to distinguish primary (early) from secondary (late) effects

• Genetic Complementation: Reintroduction of wild-type NCS6 should reverse direct effects, while indirect effects might show partial or delayed recovery

• Cross-Species Comparison: Compare the effects of Ncs6 deletion in A. niger with those observed in other fungi like M. oryzae to identify conserved direct effects

What bioinformatic approaches can predict genes most affected by loss of tRNA thiolation?

Researchers can employ several bioinformatic strategies to predict genes most affected by loss of tRNA thiolation:

• Codon Usage Analysis: Calculate the frequency of AAA/CAA/GAA codons (decoded by thiolated tRNAs) in each A. niger gene to identify those with the highest frequency

• Relative Synonymous Codon Usage (RSCU): Compare the usage of AAA vs. AAG, CAA vs. CAG, and GAA vs. GAG to identify genes with strong preference for codons decoded by thiolated tRNAs

• Functional Categorization: Perform Gene Ontology (GO) enrichment analysis of genes with high AAA/CAA/GAA frequency to identify biological processes particularly dependent on tRNA thiolation

• Expression Correlation: Analyze whether genes with high AAA/CAA/GAA content show correlated expression patterns with NCS6 across different growth conditions

• Phylogenetic Conservation: Compare codon usage patterns of orthologous genes across fungal species to identify evolutionarily conserved dependence on tRNA thiolation

• Structural RNA Analysis: Examine whether genes with high AAA/CAA/GAA frequency have particular mRNA structural features that might interact with translation efficiency

• Machine Learning Models: Develop predictive models that integrate codon usage, gene expression levels, and functional categories to predict sensitivity to tRNA thiolation loss