Recombinant Ashbya gossypii 21S rRNA pseudouridine (2819) synthase (PUS5)

What is Ashbya gossypii 21S rRNA pseudouridine(2819) synthase (PUS5)?

Ashbya gossypii PUS5 is an enzyme belonging to the pseudouridine synthase RluA family that catalyzes the isomerization of uridine to pseudouridine specifically at position 2819 in the 21S ribosomal RNA . This post-transcriptional modification is critical for RNA structure stabilization and proper ribosomal function. A. gossypii, an industrially important filamentous fungus known for riboflavin production, relies on PUS5 for optimal mitochondrial translation and subsequent metabolic functions.

How does A. gossypii PUS5 differ from its orthologs in related fungal species?

While A. gossypii shares high gene homology with Saccharomyces cerevisiae (91% of genes are syntenic), significant metabolic differences exist between these organisms, particularly in purine and pyrimidine metabolism . These metabolic distinctions suggest potential functional differences in their RNA modification enzymes, including PUS5. Unlike S. cerevisiae, which underwent whole genome duplication (WGD), A. gossypii represents a pre-WGD organism, potentially affecting its RNA modification landscape. Comparative studies indicate that pseudouridine synthase distribution varies among fungal species, with certain RNA modification enzymes present in some species but absent in others .

What is the genomic context and expression pattern of PUS5 in A. gossypii?

The PUS5 gene in A. gossypii likely maintains syntenic relationships with its S. cerevisiae ortholog, given the high degree of gene order conservation between these organisms . PUS5 encodes a mitochondrial protein responsible for 21S rRNA pseudouridylation. While specific expression data for A. gossypii PUS5 is limited, studies of similar enzymes in related fungi suggest its expression may be influenced by growth phase, carbon source availability, and stress conditions. The extensive genome re-annotation efforts for A. gossypii have improved our understanding of its metabolic pathways, providing context for PUS5 function within the organism's unique metabolism .

What expression systems are optimal for producing recombinant A. gossypii PUS5?

Several expression systems can be employed for recombinant A. gossypii PUS5 production, each with distinct advantages:

A. gossypii itself presents an interesting option as it has been demonstrated to effectively express and secrete heterologous proteins with appropriate post-translational modifications and less extensive hyperglycosylation than S. cerevisiae .

What are the most effective purification strategies for recombinant A. gossypii PUS5?

Purification of recombinant A. gossypii PUS5 typically requires a multi-step chromatographic approach:

• Affinity chromatography: Using His6-tag or other affinity tags for initial capture

• Ion-exchange chromatography: Separating charge variants and removing contaminating nucleic acids

• Size-exclusion chromatography: Final polishing step for homogeneous protein preparation

Throughout purification, maintaining reducing conditions (using DTT or β-mercaptoethanol) is crucial to preserve the catalytic cysteine residues common in pseudouridine synthases. For highest purity preparations, especially for structural studies, additional steps like heparin affinity chromatography may be beneficial due to PUS5's interaction with RNA.

What assays can accurately measure A. gossypii PUS5 enzymatic activity?

Several complementary methods can assess PUS5 activity with varying sensitivities and applications:

The choice of assay depends on the specific research question, available equipment, and whether in vitro or in vivo activity is being assessed.

How do mutations in conserved catalytic residues affect A. gossypii PUS5 activity?

Mutational analysis of conserved catalytic residues in A. gossypii PUS5 provides critical insights into structure-function relationships. The RluA family typically contains a catalytic aspartate that forms a covalent adduct with the uracil ring during the isomerization reaction. Systematic alanine scanning mutagenesis coupled with activity assays can establish the complete catalog of essential residues and their specific roles in catalysis. Comparative analysis of mutational effects between A. gossypii and S. cerevisiae PUS5 might reveal species-specific functional adaptations related to A. gossypii's distinct metabolism, particularly its capacity for riboflavin overproduction.

What structural features determine substrate recognition specificity of A. gossypii PUS5?

The substrate recognition specificity of A. gossypii PUS5 involves both sequence and structural elements of the 21S rRNA target region. Research approaches to elucidate these determinants include:

• X-ray crystallography or cryo-EM studies of PUS5-RNA complexes

• Molecular docking and MD simulations to predict binding interfaces

• SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify preferred RNA sequence/structure elements

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

These complementary approaches would reveal how PUS5 achieves its remarkable specificity for position 2819 in 21S rRNA and whether any A. gossypii-specific recognition features exist compared to orthologs in other fungi.

How does pseudouridylation at position 2819 affect ribosomal function in A. gossypii?

The impact of this specific modification extends to multiple aspects of ribosomal function:

Creating PUS5 knockout strains in A. gossypii would allow comprehensive assessment of these parameters and establish connections between this specific modification and the organism's distinctive metabolism.

What is the relationship between PUS5 activity and riboflavin production in A. gossypii?

The relationship between PUS5 activity and A. gossypii's hallmark riboflavin production represents an unexplored but intriguing research area. As a mitochondrial rRNA modification enzyme, PUS5 could influence riboflavin biosynthesis through several mechanisms:

• Mitochondrial translation efficiency: Optimizing translation of proteins involved in energy production

• Metabolic regulation: Altering redox state and energy metabolism

• Stress response integration: Coordinating RNA modification with conditions triggering riboflavin overproduction

Previous studies have highlighted connections between inositol metabolism and riboflavin production through regulatory mechanisms , suggesting that RNA modifications might similarly participate in metabolic regulatory networks. Experimental approaches would include creating PUS5 knockout or overexpression strains and assessing their riboflavin production under various conditions.

How does A. gossypii PUS5 activity change during different growth phases and stress conditions?

RNA modification enzymes often display regulated expression patterns tied to growth phases and stress responses. Comprehensive characterization of PUS5 expression and activity across:

• Developmental stages (spore, germling, vegetative mycelium, sporulation)

• Growth phases (lag, exponential, stationary)

• Stress conditions (nutrient limitation, oxidative stress, temperature shifts)

would provide insights into its regulatory context and coordination with metabolic adaptation. Of particular interest would be correlating PUS5 activity with conditions that induce riboflavin overproduction, which is significantly elevated in A. gossypii compared to other fungi .

What role does PUS5 play in A. gossypii's distinctive filamentous growth pattern?

A. gossypii's filamentous growth represents a key morphological distinction from the yeast-like growth of S. cerevisiae, despite their high genetic similarity . RNA modifications have been implicated in developmental regulation in various organisms. Investigating potential connections between mitochondrial rRNA modification by PUS5 and filamentous growth could reveal novel insights into how post-transcriptional modifications influence fungal morphogenesis. Research approaches would include microscopic analysis of growth patterns in PUS5 mutant strains under various conditions and transcriptomic profiling to identify downstream effects on genes involved in hyphal development.

How has PUS5 evolved across fungal species and what does this reveal about functional importance?

Evolutionary analysis of PUS5 across fungal species provides insights into its functional conservation and adaptation. Phylogenetic analysis coupled with selection pressure analysis (dN/dS ratios) would identify conserved domains versus rapidly evolving regions. Of particular interest would be comparing PUS5 between pre-WGD species like A. gossypii and post-WGD species like S. cerevisiae , as well as between riboflavin overproducers and non-overproducers. This evolutionary perspective could reveal how PUS5 has been maintained or adapted to support different metabolic strategies across fungal diversity.

How does the RNA modification landscape in A. gossypii compare to related fungi?

The search results mention several enzymatic differences between A. gossypii and related species . These differences likely extend to RNA modification enzymes, potentially creating a distinctive epitranscriptomic landscape in A. gossypii. Comparative pseudouridine-seq and other epitranscriptomic profiling techniques could map the complete modification landscape across these species. The absence of specific pseudouridine synthases might create compensatory modification patterns or leave certain RNA positions unmodified, potentially contributing to A. gossypii's distinctive metabolism.

This comparative approach could provide insights into how RNA modification patterns have evolved alongside metabolic diversification in fungi.

How can recombinant A. gossypii PUS5 be utilized in academic research applications?

Recombinant A. gossypii PUS5 offers several valuable applications in research:

• Tool for site-specific pseudouridylation of synthetic RNAs

• Model system for studying RNA modification mechanisms

• Target for developing RNA modification inhibitors

• Component for reconstituted in vitro translation systems

Protocols for each application would require optimization of enzyme concentration, buffer conditions, and reaction parameters to achieve efficient and specific pseudouridylation.

What are the key considerations for designing PUS5 knockout or modification studies in A. gossypii?

Creating genetic modifications in A. gossypii presents unique challenges compared to model yeasts like S. cerevisiae. Key considerations include:

• Transformation efficiency: Optimizing protocols specifically for A. gossypii

• Homologous recombination strategies: Designing appropriate flanking sequences

• Selection markers: Choosing appropriate markers given A. gossypii's resistance profile

• Verification methods: PCR, Southern blotting, and sequencing approaches

• Phenotypic analysis: Comprehensive assessment of growth, morphology, and metabolism

The filamentous growth pattern of A. gossypii may necessitate specialized approaches for transformant isolation and characterization compared to unicellular yeasts.

What experimental approaches can elucidate the interplay between RNA modifications and metabolic specialization in A. gossypii?

Investigating the relationship between RNA modifications and A. gossypii's distinctive metabolism requires integrative approaches:

• Epitranscriptome profiling under riboflavin-producing conditions

• Ribosome profiling to identify translationally regulated metabolic enzymes

• Metabolic flux analysis in RNA modification mutants

• Comparative systems biology across related fungal species

These approaches could reveal how the RNA modification machinery, including PUS5, contributes to A. gossypii's remarkable ability to overproduce riboflavin and its distinctive filamentous growth pattern despite its close evolutionary relationship to S. cerevisiae .