Recombinant Ashbya gossypii Pre-mRNA-splicing factor RSE1 (RSE1), partial

What is Ashbya gossypii and why is it important as a model organism?

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that shares a close evolutionary relationship with unicellular yeasts such as Saccharomyces cerevisiae. Its significance as a model organism stems from several key attributes: it offers a valuable system for elucidating the regulatory networks that govern functional differences between filamentous growth and yeast growth patterns . The completion of the A. gossypii genome sequence has substantially enhanced its utility as a research model . Additionally, A. gossypii has proven valuable for understanding mechanisms relevant to dimorphic yeasts such as the human pathogen Candida albicans, where morphological switching between yeast and filamentous forms is critical for virulence .

The genome structure of A. gossypii has provided definitive evidence that S. cerevisiae underwent a whole-genome duplication event during its evolution, making A. gossypii an excellent reference for studying genome evolution in yeasts . Furthermore, as a system for studying polar growth using genetic, biochemical, and genomic approaches, A. gossypii offers significant advantages compared to more complex filamentous fungi .

What is the RSE1 protein and what is its role in pre-mRNA splicing?

RSE1 (pre-mRNA-splicing factor RSE1) is a protein involved in the pre-mRNA splicing process, which is essential for gene expression in eukaryotes. While the search results don't provide specific details about RSE1's function in A. gossypii, comparative genomics indicates that it likely serves a role similar to its homologs in related yeasts . In eukaryotes generally, pre-mRNA splicing factors participate in the removal of non-coding introns from pre-mRNA, allowing the coding exons to be joined together to form mature mRNA for translation.

The RSE1 partial protein from A. gossypii is available as a recombinant protein produced in mammalian cell systems, with a purity of >85% as determined by SDS-PAGE . This recombinant form serves as a valuable research tool for studying splicing mechanisms in filamentous fungi and for comparative analyses with yeast splicing machinery.

How does the genome of A. gossypii compare to related yeast species?

The A. gossypii genome shows remarkable conservation with S. cerevisiae despite their different growth morphologies. The Ashbya Genome Database (AGD) contains 4,718 protein-encoding loci distributed across seven chromosomes . Approximately 90% of A. gossypii genes have homologs in the S. cerevisiae genome, demonstrating high genetic similarity despite their distinct morphological differences .

A. gossypii has a more compact genome with higher GC content (53%) compared to S. cerevisiae (38%) . This higher GC content occasionally presents challenges for gene annotation, particularly in assigning start codons . The genome annotation was substantially facilitated by comparative genomics, exploiting conservation of gene order and orientation (synteny) between these related genomes .

The genome sequence of A. gossypii has proven extremely useful for refining the annotation of the budding yeast genome and has provided definitive proof that S. cerevisiae underwent a whole-genome duplication event .

How does A. gossypii respond to protein secretion stress compared to other fungi?

A. gossypii exhibits an unconventional response to protein secretion stress that differs significantly from what has been observed in other yeasts and filamentous fungi. Transcriptomic analyses under recombinant protein secretion conditions and dithiothreitol-induced secretion stress revealed that A. gossypii does not activate a conventional unfolded protein response (UPR) . This is evidenced by the fact that several well-known UPR target genes (including IRE1, KAR2, HAC1, and PDI1 homologs) maintained stable expression levels under stress conditions .

Despite the absence of a conventional UPR, A. gossypii employs alternative mechanisms to manage secretion stress. These include upregulation of genes involved in:

• Protein unfolding

• Endoplasmic reticulum-associated degradation

• Proteolysis

• Vesicle trafficking

• Vacuolar protein sorting

• Secretion

• mRNA degradation

Conversely, transcription of several genes encoding secretory proteins, particularly components of the glycosylation pathway, was substantially repressed under dithiothreitol-induced stress conditions . This distinctive stress response pattern suggests that A. gossypii has evolved alternative protein quality control mechanisms that diverge from the canonical UPR observed in most other fungi.

What are the characteristics of the A. gossypii secretome and how might this impact recombinant protein production?

The secretome of A. gossypii has been characterized through both computational predictions and proteomic analyses of culture supernatants. Key findings include:

• Only 1-4% of A. gossypii proteins are likely secreted via the general secretory pathway

• Less than 33% of the secreted proteins are putative hydrolases

• Most secreted proteins have an isoelectric point between 4 and 6

• The majority of secreted proteins have a molecular mass above 25 kDa

These secretome characteristics are more similar to those of yeasts than to other filamentous fungi, which typically secrete a larger proportion of their proteome, particularly hydrolytic enzymes . This relatively modest secretory capacity might partially explain why A. gossypii has evolved alternative mechanisms to cope with secretion stress rather than relying on a conventional UPR system.

From a biotechnological perspective, understanding these secretory properties is essential for optimizing A. gossypii as a host for recombinant protein production. The atypical stress response mechanisms may require specialized strategies for engineering high-yield recombinant protein expression systems that differ from approaches used with other fungal hosts .

What structural and functional insights can be gained from studying partial recombinant RSE1?

The partial recombinant RSE1 protein available commercially provides an opportunity to investigate specific functional domains without the constraints of the full-length protein. While the search results don't specify which portion of RSE1 is represented in the partial recombinant form, typical approaches to studying partial splicing factors include:

• Domain mapping to identify regions responsible for protein-protein interactions within the spliceosome

• RNA binding assays to determine if the partial protein retains nucleic acid interaction capabilities

• In vitro splicing assays to assess whether the partial protein can complement splicing defects or act as a dominant negative

Given that A. gossypii shows unique stress response patterns compared to related fungi, investigating RSE1's role in regulated splicing under stress conditions could provide insights into alternative stress response mechanisms. The partial protein might be particularly useful for identifying critical interaction partners through pull-down assays coupled with mass spectrometry.

What are the recommended protocols for storing and reconstituting recombinant A. gossypii RSE1?

The recombinant partial RSE1 protein should be handled according to specific protocols to maintain its stability and functionality:

Storage recommendations:

• Liquid form: Stable for approximately 6 months at -20°C/-80°C

• Lyophilized form: Stable for approximately 12 months at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (preferably 50%)

• Aliquot for long-term storage at -20°C/-80°C

Following these guidelines will help ensure the protein maintains its structural integrity and functional properties for experimental applications.

What experimental approaches can be used to study RSE1 function in the context of A. gossypii biology?

To investigate RSE1 function in A. gossypii, researchers can employ several complementary approaches:

Genetic approaches:

• Gene deletion or conditional expression studies, taking advantage of A. gossypii's amenability to genetic manipulation

• CRISPR-Cas9 genome editing to introduce specific mutations in functional domains

• Promoter replacement strategies to control expression levels

Transcriptomic approaches:

• RNA-seq analysis comparing wild-type and RSE1 mutant strains to identify splicing defects

• Examination of alternative splicing patterns under different growth conditions

• Integration with high-density oligonucleotide microarrays that are becoming available for A. gossypii

Proteomic approaches:

• Immunoprecipitation coupled with mass spectrometry to identify interaction partners

• Comparative proteomics between RSE1 mutant and wild-type strains to identify downstream effects

• Analysis of protein complexes containing RSE1 under various stress conditions

Cell biological approaches:

• Subcellular localization studies using fluorescently tagged RSE1

• Analysis of nuclear organization and splicing factor dynamics

• Correlation of RSE1 localization with transcriptional activity

Researchers should consult the Ashbya Genome Database (AGD) at http://agd.unibas.ch/ for up-to-date annotation information and potential homologs in other species to guide experimental design .

How can recombinant RSE1 be used to study pre-mRNA splicing mechanisms?

Recombinant RSE1 can serve as a valuable tool for investigating splicing mechanisms through several experimental approaches:

In vitro splicing assays:

• Prepare nuclear extracts from A. gossypii or related yeasts

• Supplement extracts with recombinant RSE1 to assess functional complementation

• Use synthetic pre-mRNA substrates containing introns from genes of interest

• Analyze splicing products by gel electrophoresis and RT-PCR

Spliceosome assembly studies:

• Immobilize recombinant RSE1 on a suitable matrix

• Incubate with nuclear extracts under splicing conditions

• Isolate and identify associated spliceosomal components by mass spectrometry

• Compare complex formation under normal and stress conditions

RNA-protein interaction analyses:

• Perform electrophoretic mobility shift assays (EMSAs) with labeled pre-mRNA

• Use UV crosslinking to identify direct RNA contacts

• Employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify preferred RNA binding motifs

• Validate interactions in cellular contexts through CLIP (Cross-Linking Immunoprecipitation)

These approaches can provide insights into how RSE1 contributes to splicing efficiency and specificity in A. gossypii, potentially revealing mechanisms that differ from those in S. cerevisiae due to the unique evolutionary trajectory of these related organisms.

How does RSE1 from A. gossypii compare to homologs in S. cerevisiae and other fungi?

While the search results don't provide specific information about RSE1 sequence conservation across species, a comparative analysis framework can be established based on the general patterns observed for other proteins:

Researchers can use the Ashbya Genome Database (AGD) to identify the specific S. cerevisiae homolog of RSE1 and access relevant annotation information through the provided links to SGD (Saccharomyces Genome Database) and other databases .

How do splicing mechanisms in A. gossypii relate to its unique life cycle and morphology?

The filamentous growth pattern of A. gossypii presents distinct cellular challenges compared to unicellular yeasts, potentially requiring specialized regulation of RNA processing and gene expression. While the search results don't directly address RSE1's role in morphology, several relevant considerations can be inferred:

• A. gossypii serves as an excellent model system for studying polar growth using genetic, biochemical, and genomic approaches .

• The transition between different morphological states in related dimorphic fungi like C. albicans involves coordinated changes in gene expression patterns .

• A. gossypii exhibits unconventional stress response mechanisms that differ from those in S. cerevisiae .

These observations suggest that splicing regulation, potentially involving RSE1, might play a role in coordinating gene expression programs required for filamentous growth. Specifically, alternative splicing could generate protein isoforms optimized for different cellular compartments within the growing hyphae or for different developmental stages.

Researchers interested in this aspect could design experiments comparing splicing patterns between the growing hyphal tip and more mature regions of the mycelium, potentially revealing spatial regulation of splicing that contributes to the establishment and maintenance of filamentous morphology.

What emerging technologies could advance our understanding of RSE1 function in A. gossypii?

Several cutting-edge technologies could significantly enhance our understanding of RSE1's role in A. gossypii:

Long-read sequencing technologies:

• Direct RNA sequencing to map full-length transcripts and identify all splice variants

• Nanopore-based methods to detect RNA modifications that might influence splicing

• Long-read approaches to resolve complex splicing events and isoform diversity

Spatial transcriptomics:

• Techniques to map transcript processing events to specific cellular compartments

• Investigation of spatial heterogeneity in splicing patterns within growing hyphae

• Correlation of splicing activity with cellular differentiation in the mycelium

High-throughput mutagenesis:

• CRISPR-based screens to identify genetic interactions with RSE1

• Systematic domain mutagenesis to map functional regions of the protein

• Deep mutational scanning to comprehensively assess the impact of amino acid substitutions

Advanced imaging:

• Super-resolution microscopy to visualize spliceosome assembly and dynamics

• Live-cell imaging of fluorescently tagged splicing factors during growth and stress

• Single-molecule tracking to analyze RSE1 kinetics and interactions

Integration of these technologies with the genomic resources available through the Ashbya Genome Database could provide unprecedented insights into how RSE1 contributes to A. gossypii biology and how splicing mechanisms have evolved to support filamentous growth.

How might understanding RSE1 function contribute to biotechnological applications of A. gossypii?

A. gossypii is already industrially important for riboflavin production and has recently been explored as a host system for recombinant protein production . Understanding RSE1 function could contribute to biotechnological applications in several ways:

• Optimizing gene expression: Knowledge of splicing regulation could lead to improved gene design for heterologous expression, potentially eliminating introns that might be inefficiently processed or introducing optimized introns that enhance expression.

• Stress response engineering: Given A. gossypii's unconventional response to secretion stress , manipulating RSE1 function might help develop strains with enhanced capacity to manage the burden of recombinant protein production.

• Controlled morphology: If RSE1 influences aspects of morphological development, modulating its activity could help control fungal growth patterns during industrial fermentation, potentially optimizing biomass accumulation or protein secretion.

• Synthetic biology applications: Understanding the mechanistic details of splicing could enable the development of synthetic splicing regulators as genetic tools to control gene expression in response to specific stimuli.

These applications align with ongoing efforts to develop A. gossypii as a versatile biotechnological platform, leveraging its unique combination of yeast-like genetic tractability and filamentous growth .