Recombinant Neosartorya fumigata Pre-mRNA-splicing factor cwc24 (cwc24)

What is Neosartorya fumigata and how does it relate to Aspergillus fumigatus?

Neosartorya fumigata is a fungal species that represents the teleomorph (sexual state) of Aspergillus fumigatus, which is its anamorph (asexual state). Although historically given different names due to taxonomic conventions, recent "one name, one fungus" proposals have aimed to unify the nomenclature . N. fumigata is primarily distinguished by its ability to produce ascospores during sexual reproduction, which are heat-resistant and lenticular with two equatorial crests .

The taxonomic relationship is important for researchers because:

• The genome sequences labeled as either Neosartorya fumigata or Aspergillus fumigatus in databases may refer to the same organism

• Strain designations such as "ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100" are often used to specify the exact isolate

• Many molecular studies use these organisms interchangeably, although their protein expression patterns may differ slightly depending on life cycle stage

Unlike some related species (e.g., N. fischeri), N. fumigata is a significant opportunistic human pathogen capable of causing various forms of aspergillosis, particularly in immunocompromised individuals .

What is the function of Pre-mRNA-splicing factor cwc24 in fungal molecular biology?

The cwc24 protein is a critical component involved in pre-mRNA splicing. Based on current research, cwc24:

• Belongs to the CWC24 family of splicing factors

• Is recruited to the spliceosome during the B act complex formation

• Dissociates during the B act-to-B* transition, unlike some other splicing factors (Cwc21 and Cwc22) that remain associated through later complexes

• Plays a role in the first catalytic step of the splicing reaction

Functionally, cwc24 differs from step I splicing factors (like Cwc25 and Yju2 in S. cerevisiae) that associate with the splicing active site during the branching reaction, and from step II factors (Prp18 and Slu7) that primarily promote exon ligation .

The timing of cwc24's association with and dissociation from the spliceosome suggests it functions in preparing the spliceosome for catalytic activation rather than directly participating in the catalytic reactions themselves.

How can researchers express and purify recombinant Neosartorya fumigata cwc24?

For successful expression and purification of recombinant N. fumigata cwc24, researchers should consider the following methodological approach:

Expression Systems:

• E. coli is the preferred host system for expression, as documented in commercial recombinant products

• N-terminal tagging with 6xHis-SUMO is effective for improving solubility and facilitating purification

Purification Strategy:

• Transform expression vector containing the cwc24 gene into an appropriate E. coli strain

• Culture in LB media with appropriate antibiotic selection

• Induce protein expression with IPTG when culture reaches mid-log phase

• Harvest cells and lyse using sonication in Tris-based buffer

• Purify using nickel affinity chromatography (leveraging the His-tag)

• For higher purity applications, employ size exclusion chromatography

• Verify purity using SDS-PAGE (expected purity >90%)

• Store in Tris-based buffer with 50% glycerol for stability

The recombinant protein should be tested for functional activity through RNA binding assays or splicing reconstitution experiments to ensure proper folding.

How does cwc24 contribute to the spliceosome assembly and function?

The cwc24 protein plays a specific temporal role in spliceosome assembly and function:

• Assembly Phase: cwc24 is recruited to the spliceosome during formation of the B act complex, which occurs after the binding of the tri-snRNP (U4/U6.U5) to the pre-spliceosome (complex A)

• Functional Role: Evidence suggests cwc24 may help position the pre-mRNA within the catalytic center, potentially through interactions with both RNA and proteins

• Dissociation Timing: Unlike some persistent spliceosomal components, cwc24 dissociates during the transition from B act to B* complex, before the first catalytic step of splicing

• Protein Interactions: cwc24 likely interacts with Prp8, a highly conserved component of the U5 snRNP that anchors the splicing active site

This pattern of association and dissociation distinguishes cwc24 from:

• Core components (like U5 snRNP) that remain throughout all assembled spliceosomal complexes

• Step I factors (Cwc25 and Yju2) that associate only during the branching reaction

• Step II factors (Prp18 and Slu7) that promote exon ligation

What experimental approaches can be used to study cwc24's role in pre-mRNA splicing?

Researchers can employ several methodological approaches to investigate cwc24's specific role in pre-mRNA splicing:

In vitro Splicing Assays:

• Reconstituted splicing reactions using purified components

• Depletion-complementation experiments (deplete endogenous cwc24 and add back recombinant protein)

• RNA-protein binding studies to map interaction sites

Cellular Approaches:

• RNA-FISH (Fluorescence In Situ Hybridization) combined with immunofluorescence to visualize co-localization of cwc24 with actively spliced transcripts

• Quantitative single-cell imaging to assess recruitment of cwc24 to transcription sites

Genetic Approaches:

• CRISPR/Cas9-mediated mutations of cwc24 in model organisms

• Analysis of alternative splicing patterns in cwc24-mutant strains

Example Method from Literature (adapted from splicing factor studies):
Combined RNA-FISH and immunofluorescence microscopy has proven effective for detecting recruitment of splicing factors to specific transcription sites. This technique involves:

• Fixing cells and performing RNA-FISH using probes against the target gene

• Following with immunofluorescence using antibodies against cwc24

• Analyzing co-localization through confocal microscopy and linescan analysis

This methodology has revealed differential recruitment patterns for various splicing factors, which could be applied to understand cwc24's dynamics.

How does post-translational modification affect cwc24 function in the spliceosome?

While specific data on post-translational modifications (PTMs) of N. fumigata cwc24 is limited, research on related splicing factors suggests several potential regulatory mechanisms:

Potential PTMs affecting cwc24:

• Phosphorylation: Likely regulates timing of assembly/disassembly from the spliceosome

• Ubiquitination: May control protein turnover and availability

• SUMOylation: Could affect protein-protein interactions within the spliceosome

Methodological approaches to study PTMs:

• Mass Spectrometry Analysis:

  • Immunoprecipitate cwc24 from cellular extracts

  • Perform tryptic digestion and LC-MS/MS analysis

  • Compare modification patterns at different splicing stages

• Site-directed Mutagenesis:

  • Identify potential modification sites through sequence analysis

  • Generate mutants (e.g., S→A for phosphorylation sites)

  • Test functional consequences in splicing assays

• Phospho-specific Antibodies:

  • Develop antibodies that recognize modified forms

  • Use for Western blotting and immunofluorescence to track modification status

Understanding these modifications would provide insight into how cwc24's activity is regulated throughout the splicing cycle.

How evolutionarily conserved is cwc24 across fungal species and beyond?

The cwc24 protein belongs to the CWC24 family, which shows interesting patterns of conservation across fungal species:

Conservation patterns:

• The core function in pre-mRNA splicing appears conserved across fungi

• Sequence similarity is highest among closely related Aspergillus species

• Greater divergence is observed when comparing with distant fungal lineages

Methodological approaches to study evolutionary conservation:

• Comparative Genomics:

  • Sequence alignment of cwc24 homologs from different species

  • Identification of conserved functional domains

  • Analysis of selection pressure on different protein regions

• Functional Complementation:

  • Express cwc24 from different species in a cwc24-deficient strain

  • Assess the ability to rescue splicing defects

  • Determine which protein regions are functionally interchangeable

• Structural Biology:

  • Determine 3D structures of cwc24 from multiple species

  • Identify structurally conserved regions despite sequence divergence

This evolutionary analysis helps distinguish between core functional requirements of cwc24 and species-specific adaptations.

How does cwc24 function differ between Neosartorya fumigata and other model organisms?

The function of cwc24 shows both conservation and divergence between N. fumigata and other model organisms:

Comparison with Saccharomyces cerevisiae:

• In S. cerevisiae, Cwc24 is also involved in pre-mRNA splicing

• Both associate with the B act complex and dissociate before the first catalytic step

• Sequence homology suggests similar domain organization but with species-specific variations

Methodological approaches for comparative studies:

• Heterologous Expression:

  • Express N. fumigata cwc24 in S. cerevisiae cwc24 deletion strains

  • Assess complementation of growth and splicing phenotypes

  • Identify functional equivalence or differences

• Protein-Protein Interaction Networks:

  • Perform pull-down assays to identify interacting partners in both species

  • Compare interaction maps to identify conserved and divergent interactions

  • Use yeast two-hybrid or proximity labeling approaches

• Splicing Substrate Preferences:

  • Test activity on various pre-mRNA substrates

  • Determine if substrate specificity differs between species

Understanding these differences could reveal adaptations related to different ecological niches and pathogenicity of N. fumigata versus non-pathogenic model organisms.

What is the relationship between cwc24 and fungal pathogenicity?

While direct evidence linking cwc24 to pathogenicity is limited, several research avenues suggest potential connections:

Potential roles in pathogenicity:

• Regulation of virulence factors: Alternative splicing regulated by splicing factors like cwc24 could affect expression of virulence-related genes

• Stress adaptation: Proper pre-mRNA processing is critical for rapid adaptation to host environments

• Host-pathogen interaction: Splicing machinery may process transcripts encoding proteins involved in host immune evasion

Research approaches to investigate these connections:

• Comparative Transcriptomics:

  • Compare splicing patterns between pathogenic (N. fumigata) and non-pathogenic relatives

  • Identify alternatively spliced transcripts unique to pathogenic species

  • Determine if cwc24 regulates these pathogenicity-associated splicing events

• Infection Models:

  • Generate conditional cwc24 mutants in N. fumigata

  • Test virulence in established infection models

  • Analyze transcriptome changes during infection

• Proteomics Analysis:

  • Compare the cwc24 interaction network between infection and non-infection conditions

  • Identify infection-specific interactions that may contribute to pathogenicity

Researchers should note that N. fumigata (A. fumigatus) is a significant opportunistic pathogen that causes invasive aspergillosis, especially in immunocompromised patients, with high mortality rates .

What techniques are most effective for studying the RNA-binding properties of cwc24?

Understanding the RNA-binding properties of cwc24 is crucial for elucidating its role in splicing. Several techniques can be employed:

In vitro binding assays:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified recombinant cwc24 with labeled RNA

  • Analyze complex formation by gel electrophoresis

  • Determine binding affinity and specificity

• UV Cross-linking:

  • UV-irradiate cwc24-RNA complexes to form covalent bonds

  • Digest with RNases to identify protection patterns

  • Map binding sites through primer extension analysis

• RNA Immunoprecipitation (RIP):

  • Cross-link RNA-protein complexes in vivo

  • Immunoprecipitate cwc24 using specific antibodies

  • Identify bound RNAs through sequencing (RIP-Seq)

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

  • Expose random RNA libraries to cwc24

  • Select and amplify bound sequences

  • Identify optimal binding motifs after multiple rounds

• Surface Plasmon Resonance (SPR):

  • Immobilize either cwc24 or RNA on a sensor chip

  • Measure real-time binding kinetics

  • Determine association and dissociation rates

These methodologies can help determine if cwc24 recognizes specific RNA sequences or structural elements within the pre-mRNA, providing insights into its mechanistic role in splicing.

How can researchers investigate cwc24's interactions with other spliceosomal proteins?

Understanding protein-protein interactions is essential for deciphering cwc24's role within the spliceosome complex:

Methods for mapping protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Express tagged cwc24 in fungal cells

  • Immunoprecipitate cwc24 and associated proteins

  • Identify interacting partners by mass spectrometry

  • Verify specific interactions with Western blotting

• Yeast Two-Hybrid (Y2H):

  • Construct cwc24 fusion with DNA-binding domain

  • Screen against spliceosomal proteins fused to activation domain

  • Identify direct binary interactions

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse cwc24 to a biotin ligase (BirA*)

  • Express in cells and allow biotinylation of proximal proteins

  • Isolate biotinylated proteins and identify by mass spectrometry

• Förster Resonance Energy Transfer (FRET):

  • Tag cwc24 and potential interacting partners with fluorophore pairs

  • Measure energy transfer as indicator of proximity

  • Use in live cells to observe dynamic interactions

• Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Analyze purified complexes containing cwc24

  • Determine stoichiometry of protein complexes

  • Assess stability of different subcomplexes

These approaches would help establish cwc24's position within the intricate network of interactions that drive spliceosome assembly and function, particularly focusing on its associations with proteins like Prp8, which anchors the splicing active site .