Recombinant Neurospora crassa Pre-mRNA-splicing factor cwc-24 (cwc-24)

What is the cwc-24 protein and what is its primary function?

Cwc24 (Complexed with Cef1 protein 24) is an essential pre-mRNA splicing factor originally identified as a component of the NTC (Nineteen Complex) in Saccharomyces cerevisiae and has orthologs in many organisms including Neurospora crassa. The primary function of cwc-24 is to facilitate pre-mRNA splicing by participating in spliceosome assembly and activation .

In Neurospora crassa specifically, cwc-24 is a 405 amino acid protein with a molecular mass of approximately 44.4 kDa that belongs to the CWC24 family . The protein is involved in orchestrating the organization of the spliceosome into an active configuration prior to Prp2-mediated spliceosome remodeling and in promoting specific interactions of U5 and U6 snRNAs with the 5′ splice site for accurate 5′ splice site selection .

Why is Neurospora crassa used as a model organism for studying cwc-24 and splicing?

Neurospora crassa is an excellent model organism for studying cwc-24 and pre-mRNA splicing for several reasons:

• Historical importance: Neurospora has been used in scientific research since 1843 and was instrumental in establishing the field of microbial genetics. Experiments with Neurospora "inspired the development of microbial genetics and initiated the molecular revolution in biology by demonstrating that genes encode enzymes" .

• Safety profile: N. crassa is considered safe for laboratory use with no significant pathogenic or allergenic effects. It has no known production of dangerous secondary metabolites, making it suitable for academic and commercial research .

• Genetic resources: Over 1,000 loci have been mapped on the chromosomes of Neurospora crassa, and its genome has been completely sequenced, providing a comprehensive genetic framework for research .

• Evolutionary conservation: The splicing machinery, including cwc-24, is evolutionarily conserved across eukaryotes, making findings in Neurospora potentially applicable to other organisms, including humans. For instance, the human ortholog of cwc-24, RNF113A, is associated with the disorder trichothiodystrophy .

How does the depletion of cwc-24 affect splicing efficiency in different transcripts?

The depletion of cwc-24 has significant but variable effects on the splicing of different transcripts, as demonstrated by both in vitro and in vivo studies:

In vitro splicing effects of cwc-24 depletion:

The data demonstrates that cwc-24 is a general splicing factor required for the efficient splicing of multiple transcripts, but its requirement is especially critical for pre-U3, which contains a non-canonical branchpoint sequence (GACUAAC instead of the consensus UACUAAC) .

In vivo effects of cwc-24 depletion:

These results confirm that cwc-24 depletion significantly affects splicing of transcripts with non-consensus branchpoint sequences in vivo as well .

What is the molecular mechanism by which cwc-24 facilitates spliceosome assembly?

Cwc24 facilitates spliceosome assembly through several molecular mechanisms:

• U2 snRNP stabilization: Cwc24 is crucial for the stable association of U2 snRNP with pre-mRNAs. Depletion of cwc-24 results in reduced levels of U2 snRNA in splicing complexes, suggesting a weaker association of this snRNA with the spliceosome .

• snRNP interactions: Co-immunoprecipitation experiments have shown that cwc-24 significantly co-precipitates with the snRNAs U2, U6, and U4, suggesting its involvement in the early stages of spliceosome assembly before activation .

• 5′ splice site positioning: Cwc24 binds directly to pre-mRNA at the 5′ splice site, spanning the 5′ splice junction. This binding is critical for proper positioning of U5 and U6 snRNAs at the 5′ splice site during Prp2-mediated spliceosome remodeling .

• Regulation of Prp19 association: Cwc24 affects the association of Prp19p with splicing complexes. TAP-Prp19 extracts depleted of cwc-24 showed a reduction in the levels of U2 and U6 snRNAs compared to mock-depleted extracts .

• ZF-domain functionality: The zinc finger domain of cwc-24 is essential for its stable association with the spliceosome. Mutations in this domain (e.g., C144A) affect the interaction of cwc-24 with pre-mRNA, leading to aberrant U5–5′SS and U6–5′SS interactions .

What experimental approaches are most effective for studying cwc-24 function in splicing?

Several effective experimental approaches have been employed to study cwc-24 function in splicing:

• Immunodepletion and complementation assays: Antibodies against recombinant full-length cwc-24 protein can be used to deplete the protein from splicing extracts. Adding back recombinant cwc-24 to the depleted extracts restores splicing activity, allowing for the study of various cwc-24 mutants .

• In vitro splicing assays: Using different pre-mRNA substrates (pre-U3, pre-ACT1, pre-TEF4) with cwc-24-depleted extracts to assess the role of cwc-24 in splicing various transcripts .

• Branchpoint mutation analysis: Creating mutations in the branchpoint sequence (e.g., changing non-consensus to consensus sequences) to study how cwc-24 facilitates splicing of transcripts with non-canonical features .

• UV-crosslinking: This technique is effective for studying the direct interaction of cwc-24 with pre-mRNA. Pre-mRNA can be labeled with 32P for detection of crosslinks .

• Co-immunoprecipitation of splicing complexes: TAP-tagged proteins (e.g., TAP-Prp19p) can be used to pull down splicing complexes from cwc-24-depleted extracts to study the effects on spliceosome composition .

• Genetic depletion systems: Systems like Δcwc24/GAL-CWC24 allow for controlled depletion of cwc-24 in vivo by switching from galactose to glucose medium, enabling the study of long-term effects of cwc-24 depletion .

• RT-qPCR analysis: This method can be used to quantify the levels of mature, precursor, and total RNAs after cwc-24 depletion, providing insights into splicing efficiency in vivo .

How does the zinc finger domain of cwc-24 contribute to splicing fidelity?

The zinc finger (ZF) domain of cwc-24 plays a crucial role in maintaining splicing fidelity through several mechanisms:

• Spliceosome association: The ZF-motif is required for stable association of cwc-24 with the spliceosome. Mutants lacking the ZF domain or containing mutations in the zinc-binding site (e.g., C144A) show limited or no binding to the spliceosome .

• Pre-mRNA interaction: The C144A mutation in the ZF domain affects the interaction of cwc-24 with pre-mRNA, leading to aberrant U5–5′SS and U6–5′SS interactions .

• Prp8 positioning: The interaction of Prp8 (a key component of the U5 snRNP) with the 5′ splice site is less specific in the absence of cwc-24 or with cwc-24 carrying the C144A mutation .

• 5′ splice site selection: When cwc-24 binds to the 5′ splice site through its ZF domain, it prevents Prp8 from interacting with the 5′ splice site and allows proper positioning of U5 and U6 snRNAs during Prp2-mediated spliceosome remodeling .

• Prevention of aberrant cleavage: In the absence of cwc-24, splicing occurs with very low efficiency, and a fraction of pre-mRNA is aberrantly cleaved at the -5 position of the 5′ exon .

Research has demonstrated that full-length cwc-24 and the N1 mutant (which retains the ZF domain) bind pre-catalytic spliceosomes to saturation levels at 3.5 nM, whereas mutants lacking the ZF domain showed limited or no binding even at concentrations up to 350 nM .

What are the optimal conditions for expressing and purifying recombinant Neurospora crassa cwc-24?

Based on the available research, the following protocols and conditions are recommended for the expression and purification of recombinant Neurospora crassa cwc-24:

Expression System:

• E. coli is commonly used for recombinant expression of cwc-24

• BL21(DE3) strain is preferred for high-level expression

• Induction with 0.5 mM IPTG at 18°C for 16-20 hours minimizes inclusion body formation

Vector and Tags:

• pGEX vectors for GST-tagged cwc-24 expression

• pET vectors for His-tagged cwc-24 expression

• The GST tag has been successfully used for functional complementation studies

Purification Strategy:

• For GST-tagged cwc-24:

  • Lysis in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and protease inhibitors

  • Affinity purification using glutathione-Sepharose beads

  • Elution with reduced glutathione (10-20 mM)

  • Optional tag removal using PreScission protease

• For functional studies:

  • Recombinant GST-Cwc24p at 1 μM concentration has been shown to restore splicing activity in depleted extracts

Quality Control:

• SDS-PAGE analysis to confirm purity and integrity

• Western blotting with anti-cwc-24 antibodies

• Functional testing in in vitro splicing assays

How can one effectively deplete cwc-24 from splicing extracts for functional studies?

Effective depletion of cwc-24 from splicing extracts is crucial for functional studies. The following methods have been successfully employed:

• Antibody-based immunodepletion:

  • Raise antibodies against recombinant full-length cwc-24 protein

  • Couple anti-cwc-24 antibodies to protein A-sepharose beads

  • Incubate splicing extracts with the antibody-coupled beads

  • Confirm depletion by Western blot analysis

  • This method has been shown to effectively deplete cwc-24 with no detectable protein remaining

• Genetic depletion systems:

  • Use strains like Δcwc24/GAL-CWC24, where cwc-24 expression is under the control of a galactose-inducible promoter

  • Shift cells from galactose to glucose medium to repress cwc-24 expression

  • Grow cells in glucose for up to 48 hours for complete depletion

  • Prepare splicing extracts from these cells

  • This method allows for studying the long-term effects of cwc-24 depletion in vivo

• Metabolic depletion:

  • As described in Wu et al. (2007), metabolic depletion can be used to remove cwc-24 from extracts

  • This approach may offer advantages for certain experimental setups

• Validation of depletion:

  • Western blotting is the primary method to confirm cwc-24 depletion

  • Functional testing using in vitro splicing assays with known cwc-24-dependent substrates (e.g., pre-U3)

  • RT-qPCR to analyze the effects on splicing in vivo

What experimental design is optimal for studying cwc-24's role in non-canonical branchpoint recognition?

An optimal experimental design for studying cwc-24's role in non-canonical branchpoint recognition would include:

• Substrate selection and design:

  • Use pre-U3 RNA, which contains a non-canonical branchpoint sequence (GACUAAC)

  • Create mutant versions with consensus branchpoint sequences (UACUAAC) as controls

  • Design additional substrates with various non-canonical branchpoint sequences to test specificity

  • Include pre-TEF4 and pre-ACT1 as controls with canonical branchpoint sequences

• Depletion and complementation assays:

  • Deplete cwc-24 from splicing extracts using antibody-based immunodepletion

  • Complement with recombinant wild-type cwc-24

  • Complement with cwc-24 mutants (especially zinc finger domain mutants)

  • Measure splicing efficiency for each substrate under each condition

• Analysis of spliceosome assembly:

  • Use native gel electrophoresis to analyze the formation of splicing complexes

  • Perform RNA analysis to determine the levels of U2 snRNA in splicing complexes

  • Conduct co-immunoprecipitation experiments to study the association of U2 snRNP components with the spliceosome

• Direct binding studies:

  • Use UV-crosslinking to study the direct interaction of cwc-24 with pre-mRNA at the branchpoint region

  • Compare crosslinking patterns between canonical and non-canonical branchpoint sequences

• In vivo validation:

  • Use RT-qPCR to analyze the effects of cwc-24 depletion on splicing of transcripts with non-canonical branchpoint sequences

  • Compare the effects on transcripts with canonical branchpoint sequences

  • Measure mature, precursor, and total RNA levels to assess splicing efficiency

This comprehensive approach would provide insights into how cwc-24 facilitates the recognition and utilization of non-canonical branchpoint sequences in pre-mRNA splicing.

How can researchers investigate the interactions between cwc-24 and other splicing factors?

Researchers can employ several complementary approaches to investigate the interactions between cwc-24 and other splicing factors:

• Co-immunoprecipitation (Co-IP):

  • Use tagged versions of cwc-24 (e.g., TAP-cwc-24, ProtA-cwc-24) to pull down interacting proteins

  • Perform reciprocal Co-IP with tagged versions of suspected interacting partners

  • Analyze the precipitated proteins by Western blotting or mass spectrometry

  • This approach has successfully identified interactions between cwc-24 and Cef1p, as well as Brr2p

• Yeast two-hybrid assays:

  • Test direct protein-protein interactions between cwc-24 and other splicing factors

  • Map interaction domains using truncated versions of the proteins

  • This approach can help identify direct physical interactions

• Immunodepletion and add-back experiments:

  • Deplete cwc-24 from extracts and assess the effect on other splicing factors

  • For example, TAP-Prp19 extracts depleted of cwc-24 showed reduced levels of U2 and U6 snRNAs

  • Add back cwc-24 to restore normal interactions

• snRNP co-immunoprecipitation:

  • Use ProtA-cwc-24 to identify which snRNPs associate with cwc-24

  • Extract RNAs from the immunoprecipitates and analyze by RT-qPCR using specific primers for each snRNA

  • This approach revealed that cwc-24 significantly co-precipitates with snRNAs U2, U6, and U4

• Crosslinking and mass spectrometry:

  • Use chemical crosslinking to capture transient interactions

  • Analyze crosslinked complexes by mass spectrometry to identify interacting partners

  • This approach has been used to identify cwc-24 as a component of the NTC complex

• Genetic interaction studies:

  • Create conditional mutants of cwc-24 and potential interacting partners

  • Look for synthetic lethality or other genetic interactions

  • This approach can reveal functional relationships between proteins

• Structural studies:

  • Use NMR or X-ray crystallography to determine the structure of cwc-24 in complex with interacting partners

  • This can provide detailed information about the molecular basis of these interactions

These methodologies, used in combination, can provide a comprehensive understanding of how cwc-24 interacts with other splicing factors and how these interactions contribute to spliceosome assembly and function.

What are the implications of cwc-24 dysfunction for human disease?

While most research on cwc-24 has focused on its role in model organisms like yeast and Neurospora, there are significant implications for human disease:

How can research on cwc-24 contribute to understanding splicing-related diseases?

Research on cwc-24 can significantly contribute to understanding splicing-related diseases through several avenues:

• Mechanistic insights:

  • Studies of cwc-24 provide fundamental insights into the mechanisms of spliceosome assembly and activation.

  • Understanding how cwc-24 facilitates the recognition of non-canonical branchpoint sequences may illuminate how splicing mutations lead to disease.

  • The role of cwc-24 in promoting specific interactions of U5 and U6 with the 5′ splice site has implications for understanding how splice site mutations affect splicing fidelity .

• Model for studying splicing fidelity:

  • Cwc-24's role in preventing aberrant 5′ splice site cleavage makes it a valuable model for studying how splicing fidelity is maintained .

  • This can inform our understanding of diseases caused by cryptic splice site activation.

• Therapeutic target identification:

  • Identifying the networks of interactions involving cwc-24 and its human ortholog RNF113A may reveal new therapeutic targets for splicing-related diseases.

  • The zinc finger domain of cwc-24, which is critical for its function, represents a potential target for therapeutic intervention .

• Biomarker development:

  • Understanding the splicing defects caused by cwc-24 dysfunction could lead to the development of biomarkers for early detection of splicing-related diseases.

  • These biomarkers could include specific splicing patterns or aberrant splice products.

• Personalized medicine approaches:

  • Knowledge of how cwc-24 facilitates splicing of transcripts with non-canonical features can inform personalized medicine approaches for patients with splicing mutations.

  • This could lead to therapies tailored to specific splicing defects.

By advancing our understanding of the fundamental mechanisms of pre-mRNA splicing, research on cwc-24 contributes to a broader framework for addressing splicing-related diseases through targeted therapeutic interventions.

What are the most promising avenues for future research on cwc-24?

Several promising avenues for future research on cwc-24 include:

• Structural studies:

  • Determining the three-dimensional structure of cwc-24, particularly in complex with pre-mRNA and other splicing factors

  • Using cryo-electron microscopy to visualize cwc-24 within the context of the spliceosome

  • These studies would provide insights into the molecular mechanisms of cwc-24 function

• Comparative analysis across species:

  • Expanding studies to compare the function of cwc-24 orthologs across different species, including humans (RNF113A)

  • Investigating whether the role of cwc-24 in non-canonical branchpoint recognition is conserved in higher eukaryotes

  • This would enhance our understanding of the evolutionary conservation of splicing mechanisms

• Global analysis of cwc-24-dependent splicing events:

  • Using RNA-seq and other high-throughput approaches to identify all transcripts whose splicing depends on cwc-24

  • Characterizing the features that make certain transcripts more dependent on cwc-24 than others

  • This would provide a comprehensive view of cwc-24's role in the splicing network

• Dynamic interactions during spliceosome assembly:

  • Using single-molecule techniques to study the dynamics of cwc-24 association and dissociation during spliceosome assembly

  • Investigating the timing and mechanism of cwc-24 displacement during Prp2-mediated spliceosome remodeling

  • This would enhance our understanding of the temporal regulation of splicing

• Therapeutic applications:

  • Developing small molecules that can modulate cwc-24 function or mimic its activity

  • Exploring the potential of cwc-24-based approaches for correcting splicing defects in disease

  • This could lead to novel therapeutic strategies for splicing-related disorders

• Integration with other cellular processes:

  • Investigating how cwc-24 function is integrated with other cellular processes, such as transcription and mRNA export

  • Studying how cwc-24 activity is regulated in response to cellular stress or developmental cues

  • This would provide insights into the broader biological context of cwc-24 function

How might CRISPR-Cas9 technology be applied to study cwc-24 function?

CRISPR-Cas9 technology offers powerful approaches for studying cwc-24 function:

• Generation of cwc-24 mutants:

  • Creating precise mutations in the cwc-24 gene, particularly in the zinc finger and RING finger domains

  • Introducing mutations that mimic those found in human diseases associated with RNF113A

  • This would allow for detailed structure-function analysis

• Conditional knockout systems:

  • Developing inducible CRISPR systems to deplete cwc-24 with temporal control

  • Using tissue-specific promoters to study cwc-24 function in specific cell types

  • This would enable the study of acute and tissue-specific effects of cwc-24 depletion

• Tagged versions for live imaging:

  • Inserting fluorescent tags into the endogenous cwc-24 locus to visualize its subcellular localization and dynamics in living cells

  • Creating split fluorescent protein systems to visualize interactions between cwc-24 and other splicing factors in real-time

  • This would provide insights into the spatial and temporal dynamics of cwc-24 function

• High-throughput screening:

  • Using CRISPR-based screens to identify genetic interactions with cwc-24

  • Identifying synthetic lethal or suppressor interactions that provide insights into cwc-24 function

  • This would place cwc-24 in a broader genetic network

• Base editing applications:

  • Using CRISPR base editors to introduce specific point mutations in cwc-24, such as the C144A mutation in the zinc finger domain

  • This would allow for precise manipulation of cwc-24 function without creating double-strand breaks

• Therapeutic proof-of-concept studies:

  • Using CRISPR to correct mutations in RNF113A in patient-derived cells

  • Testing whether restoration of RNF113A function can rescue splicing defects

  • This would provide proof-of-principle for gene therapy approaches targeting RNF113A-related disorders

These CRISPR-based approaches would complement traditional biochemical and genetic methods, providing new insights into cwc-24 function and potentially leading to therapeutic applications.

What are common challenges in working with recombinant cwc-24 and how can they be addressed?

Researchers working with recombinant cwc-24 may encounter several challenges:

• Protein solubility issues:

  • Challenge: Cwc-24 may form inclusion bodies during expression due to its zinc finger and RING finger domains

  • Solution: Express at lower temperatures (16-18°C), use solubility-enhancing tags (MBP, SUMO), or add zinc to the growth medium and purification buffers to stabilize the zinc finger domain

• Maintaining protein activity:

  • Challenge: Loss of zinc from the zinc finger domain can impair cwc-24 function

  • Solution: Include zinc in purification buffers, avoid strong chelating agents, use reducing agents to prevent oxidation of cysteines, and store the protein with glycerol at -80°C

• Protein degradation:

  • Challenge: Cwc-24 may be susceptible to proteolytic degradation

  • Solution: Use protease inhibitors during purification, work at 4°C, minimize freeze-thaw cycles, and consider adding stabilizing agents like glycerol or trehalose

• Functional assay limitations:

  • Challenge: Assessing cwc-24 function requires complex in vitro splicing assays

  • Solution: Develop simplified assays focusing on specific aspects of cwc-24 function, such as RNA binding or protein-protein interactions

• Antibody specificity:

  • Challenge: Obtaining specific antibodies for cwc-24 immunodepletion and Western blotting

  • Solution: Use recombinant full-length cwc-24 as an immunogen, purify antibodies by affinity chromatography, and validate specificity using cwc-24-depleted extracts as controls

• Reproducibility in splicing assays:

  • Challenge: Variability in splicing efficiency across different extract preparations

  • Solution: Standardize extract preparation methods, include internal controls, and perform multiple biological replicates

How can inconsistencies in cwc-24 experimental data be reconciled?

Researchers may encounter inconsistencies in cwc-24 experimental data, which can be reconciled through several approaches:

• Differences between in vitro and in vivo results:

  • Inconsistency: Effects of cwc-24 depletion on splicing may differ between in vitro and in vivo systems

  • Reconciliation: Consider differences in depletion efficiency, timing, and cellular context. For example, in vivo depletion over 8 hours showed milder effects on non-U3 transcripts compared to in vitro immunodepletion

• Variability in substrate dependency:

  • Inconsistency: Some studies may show stronger effects on specific substrates than others

  • Reconciliation: Carefully analyze substrate features (branchpoint sequences, intron length, secondary structures) and experimental conditions. Different substrates may have varying dependencies on cwc-24

• Species-specific differences:

  • Inconsistency: Results from yeast and Neurospora studies may not align perfectly

  • Reconciliation: Consider evolutionary differences in splicing mechanisms and perform comparative studies to identify conserved and divergent aspects of cwc-24 function

• Methodological variations:

  • Inconsistency: Different depletion methods may yield different results

  • Reconciliation: Compare immunodepletion, genetic depletion, and metabolic depletion approaches side-by-side, and consider combining multiple methods for validation

• Domain-specific functions:

  • Inconsistency: Studies focusing on different domains of cwc-24 may yield apparently contradictory results

  • Reconciliation: Recognize that different domains (e.g., zinc finger vs. RING finger) may have distinct functions, and design experiments to test domain-specific hypotheses

• Context-dependent interactions:

  • Inconsistency: Cwc-24 interactions with other factors may vary across studies

  • Reconciliation: Consider the dynamic nature of spliceosome assembly and the possibility that cwc-24 interactions change during the splicing cycle