Recombinant Neurospora crassa Protein VTS1 (vts-1), partial

What is VTS1 in Neurospora crassa and what are its primary functions?

VTS1 in Neurospora crassa is a sequence- and structure-specific RNA-binding protein that likely functions in post-transcriptional regulation of specific mRNAs. Based on its homologs in other fungi, VTS1 contains a sterile-α motif (SAM) domain that mediates RNA binding to specific structural elements . In Saccharomyces cerevisiae, the VTS1 homolog has been implicated in Okazaki fragment processing by modulating the endonuclease activity of Dna2, suggesting a role in DNA replication and repair . While specific functions in N. crassa are still being characterized, it may be involved in regulating hyphal growth, cell wall integrity, or other processes essential for fungal development.

The protein likely performs several key functions:

• Binding to specific RNA sequences/structures to regulate their processing, localization, or translation

• Potentially interacting with the COT-1 pathway components which regulate polar hyphal growth

• Possible involvement in cell wall remodeling pathways similar to Ssd1 in yeast

How does VTS1 in N. crassa compare structurally and functionally to homologs in other fungi?

In S. cerevisiae, Vts1 functions in:

• Post-transcriptional regulation of specific mRNAs containing its binding site at their 3'-untranslated region

• Okazaki fragment processing through interaction with Dna2 endonuclease

• The protein was originally identified as a multi-copy suppressor of vti1-2 mutant cells with defects in growth and vacuole transport

In N. crassa, VTS1 likely has adapted to filamentous fungal physiology, potentially interacting with:

• The COT-1 pathway, which regulates polar hyphal growth and cell wall remodeling

• Cell wall integrity mechanisms, which in filamentous fungi must account for continuous hyphal extension

• RNA regulation networks specific to N. crassa's complex developmental stages including conidiation

What expression patterns does vts-1 exhibit during different developmental stages?

While the search results don't provide direct expression data for vts-1 in N. crassa across developmental stages, its functions can be inferred by examining related regulatory systems. Based on the potential involvement in hyphal growth regulation and cell wall remodeling:

• Expression likely fluctuates throughout the N. crassa life cycle, with potential upregulation during active hyphal extension phases

• May show increased expression during conditions that require substantial cell wall remodeling

• Could be co-regulated with components of the COT-1 pathway genes, which are critical for proper hyphal growth

For precise expression data, RNA-seq analysis across different developmental stages (germination, hyphal growth, conidiation, and sexual development) would be necessary. Researchers should consider examining vts-1 expression in comparison with known developmental markers in N. crassa.

What are the recommended methods for recombinant expression and purification of N. crassa VTS1?

Based on established protocols for similar RNA-binding proteins, the following optimized methodology is recommended for recombinant VTS1 expression:

Expression system:

• Clone the N. crassa vts-1 open reading frame into a pET28 vector with an N-terminal 6×His tag

• Transform into E. coli BL21 (CodonPlus) strain for efficient expression

• Culture cells at 25°C until A600 reaches 0.5, then induce with 0.1 mM IPTG for 4 hours

Purification protocol:

• Harvest cells by centrifugation and resuspend in buffer T500 containing:

  • 50 mM Tris–HCl, pH 8.0

  • 500 mM NaCl

  • 10% glycerol

  • 0.1% Nonidet P-40 (NP-40)

  • Protease inhibitors (1 mM PMSF, 0.1 mM benzamidine, 1.25 μg/ml leupeptin, 0.625 μg/ml pepstatin A)

• Lyse cells using sonication

• Purify using Ni-NTA affinity chromatography

• Apply additional purification steps (ion exchange, size exclusion) to achieve high purity

• Store in buffer containing 20% glycerol at -80°C to maintain protein stability

Key considerations:

• RNA-binding proteins often co-purify with bacterial RNA; include RNase treatment if RNA-free protein is required

• The SAM domain requires proper folding for RNA-binding activity; avoid harsh denaturation conditions

• Include reducing agents to prevent disulfide bond formation

How can researchers effectively study the RNA-binding specificity of VTS1?

To comprehensively characterize VTS1 RNA-binding specificity, researchers should employ multiple complementary approaches:

RNA target identification:

• RNA Immunoprecipitation (RIP) - Pull down VTS1-bound RNAs from N. crassa cells expressing tagged VTS1

• Cross-Linking and Immunoprecipitation (CLIP) - Provides higher resolution of binding sites through UV cross-linking

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) - Identifies optimal binding motifs from random RNA pools

Binding characterization:

• Electrophoretic Mobility Shift Assays (EMSA) - Determine binding affinity and specificity

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) - Measure kinetics of RNA-protein interactions

• Fluorescence Anisotropy - Quantify binding in solution

Structural studies:

• X-ray crystallography or NMR of the SAM domain bound to target RNA sequences

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Mutational analysis of key residues in the SAM domain to determine critical binding interactions

When designing RNA substrates for binding studies, researchers should consider both sequence and structural elements, as the SAM domain typically recognizes specific RNA structural motifs rather than just primary sequences .

How might VTS1 interact with the COT-1 pathway in N. crassa?

The potential interaction between VTS1 and the COT-1 pathway represents an important area for investigation, given the critical role of COT-1 in regulating polar hyphal growth in N. crassa .

Possible mechanisms of interaction:

• Post-transcriptional regulation: VTS1 might bind to mRNAs encoding components of the COT-1 pathway, regulating their translation or stability. This could create an additional layer of control over this essential signaling cascade.

• Direct protein interaction: Similar to how SKB1 physically interacts with COT1 (as shown through co-immunoprecipitation experiments ), VTS1 might directly interact with COT-1 or other pathway components.

• Shared regulatory targets: Both VTS1 and the COT-1 pathway might regulate overlapping sets of genes involved in hyphal growth and cell wall remodeling.

Experimental approaches to investigate this interaction:

• Co-immunoprecipitation using tagged versions of VTS1 and COT-1 pathway proteins

• RNA-seq analysis comparing wild-type and vts-1 deletion strains to identify effects on COT-1 pathway gene expression

• Genetic interaction studies examining phenotypes of double mutants (vts-1 deletion with cot-1 or gul-1 mutations)

• Localization studies to determine if VTS1 co-localizes with COT-1 pathway components in specific cellular compartments

The NDR kinase COT1 has been shown to undergo arginine methylation , suggesting that post-translational modifications play important roles in this pathway, potentially providing additional regulatory mechanisms that might involve VTS1.

What role might VTS1 play in cell wall integrity in N. crassa?

Cell wall integrity is crucial for filamentous fungi, and several lines of evidence suggest VTS1 may be involved in this process:

• In yeast, the RNA-binding protein Ssd1 functions in the Cell Wall Integrity (CWI) pathway . If VTS1 shares functional similarities with Ssd1, it might similarly regulate cell wall-related processes in N. crassa.

• The COT-1 pathway, which may interact with VTS1, is known to regulate cell wall remodeling in N. crassa .

• Deletion of protein arginine methyltransferases (PRMTs) in N. crassa affects susceptibility to cell wall-targeting compounds: Δamt-1 shows increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole, while Δskb-1 exhibits increased tolerance to the chitin synthase inhibitor polyoxin D .

Experimental approaches to investigate this role:

• Phenotypic analysis: Test vts-1 deletion or overexpression strains for altered sensitivity to cell wall-disturbing agents (Congo Red, Calcofluor White, polyoxin D, voriconazole).

• Transcriptome analysis: Identify cell wall-related genes whose expression is altered in vts-1 mutants.

• RIP-seq analysis: Determine if VTS1 directly binds to mRNAs encoding cell wall biosynthesis enzymes or signaling components.

• Genetic interaction studies: Examine epistatic relationships between vts-1 and known cell wall integrity genes.

• Cell wall composition analysis: Compare the chitin, glucan, and other polysaccharide content between wild-type and vts-1 mutant strains.

How do post-translational modifications affect VTS1 function?

Post-translational modifications likely play crucial roles in regulating VTS1 function, particularly considering the importance of such modifications in related regulatory systems in N. crassa:

Potential modifications of VTS1:

• Arginine methylation: The protein arginine methyltransferases (PRMTs) in N. crassa (AMT-1, AMT-3, and SKB-1) affect various aspects of fungal growth and development . These enzymes might modify VTS1 to regulate its activity, localization, or interactions.

• Phosphorylation: Given the interaction between SKB1 and the NDR kinase COT1 in N. crassa , phosphorylation might similarly regulate VTS1 function, potentially in a manner coordinated with arginine methylation.

• Other modifications: Ubiquitination, SUMOylation, or acetylation could further modulate VTS1 stability or activity.

Methods to identify and characterize these modifications:

• Mass spectrometry analysis of purified VTS1 to identify modification sites

• Mutation of putative modification sites to assess functional consequences

• In vitro modification assays using purified enzymes (PRMTs, kinases)

• Immunoprecipitation with modification-specific antibodies

• Comparative analysis of VTS1 modifications under different growth conditions

The interplay between different types of modifications (e.g., arginine methylation and phosphorylation) may create complex regulatory networks controlling VTS1 function in response to various environmental and developmental signals .

What are the challenges in generating and studying vts-1 deletion strains in N. crassa?

Creating and analyzing vts-1 deletion strains presents several technical challenges that researchers should anticipate:

Strain construction challenges:

• If vts-1 is essential, complete deletion may be lethal, requiring conditional knockdown approaches

• Potential compensatory mechanisms through related RNA-binding proteins might mask phenotypes

• The high rate of homologous recombination in N. crassa while advantageous for gene targeting, requires careful design of deletion constructs

Phenotypic analysis considerations:

• Subtle growth or developmental phenotypes may require specialized assays beyond standard growth rate measurements

• Environment-dependent phenotypes might only manifest under specific stress conditions

• Changes in branching patterns should be quantitatively analyzed, as seen with other regulatory mutants like Δamt-3, where distances between branches were significantly longer than wild type

Recommended approaches:

• Generate multiple independent knockout strains to confirm phenotypes

• Use C-terminal tagging strategies before attempting complete deletion to assess protein localization

• Consider creating point mutations in functional domains rather than complete deletions

• Employ inducible RNAi systems if deletion proves lethal

• Analyze phenotypes under diverse growth conditions (different media, temperatures, stressors)

What techniques are most effective for studying VTS1 protein-protein interactions?

Understanding VTS1's interaction network is crucial for elucidating its functions. Several complementary techniques are recommended:

In vivo interaction studies:

• Co-immunoprecipitation with MYC-tagged proteins: This approach was successfully used to demonstrate interaction between SKB1 and COT1 in N. crassa . Similar tagging of VTS1 could identify its binding partners.

• Proximity-dependent biotin identification (BioID): Fusing VTS1 to a biotin ligase allows biotinylation of proximal proteins, which can then be purified and identified by mass spectrometry.

• Yeast two-hybrid screening: While not performed in N. crassa directly, this can identify potential interactors that can then be verified in N. crassa.

In vitro interaction studies:

• Pull-down assays with recombinant proteins: Express and purify VTS1 with affinity tags to identify direct binding partners.

• Surface plasmon resonance or bio-layer interferometry: Quantify binding kinetics between VTS1 and potential interacting proteins.

• Protein microarrays: Screen for interactions with many potential partners simultaneously.

Genetic interaction studies:

• Create double mutants of vts-1 with genes encoding potential interacting proteins

• Look for synthetic phenotypes indicating functional relationships

• Perform suppressor screens to identify genes that when mutated can compensate for vts-1 deletion

The physical interaction between SKB1 and COT1 demonstrated by co-immunoprecipitation provides a methodological template for similar studies with VTS1.

How can studying VTS1 contribute to understanding fungal growth regulation?

Research on VTS1 has significant potential to advance our understanding of fungal growth regulation mechanisms:

• Integration of post-transcriptional control with kinase signaling pathways: VTS1 may represent an important link between RNA regulation and signaling cascades like the COT-1 pathway , providing insight into how these regulatory systems are coordinated.

• Cell wall remodeling mechanisms: Understanding VTS1's potential role in cell wall integrity could reveal novel regulatory mechanisms controlling fungal morphogenesis and adaptation to environmental stress.

• Evolutionary adaptations in filamentous fungi: Comparing VTS1 functions between filamentous fungi and yeasts can illuminate how RNA-binding proteins have evolved specifically for filamentous growth regulation.

• Model for studying RNA-binding protein specialization: N. crassa VTS1 provides an excellent model for studying how RNA-binding proteins acquire specialized functions in different organisms despite conserved core domains.

Future research directions should include comprehensive identification of VTS1 RNA targets in N. crassa and characterization of how these targets differ from those of homologous proteins in other fungi, potentially revealing fundamental principles of post-transcriptional regulation in fungal development.

What are the most promising future research directions for VTS1 in N. crassa?

Several high-priority research directions for VTS1 in N. crassa would significantly advance our understanding of this protein:

• Comprehensive RNA target identification: Apply transcriptome-wide approaches like CLIP-seq to identify all RNA targets of VTS1, creating a "VTS1 regulon" map.

• Structure-function analysis: Determine the crystal structure of the N. crassa VTS1 SAM domain, ideally in complex with target RNA, to understand binding specificity.

• Integration with signaling networks: Map the relationships between VTS1 and established signaling pathways, particularly the COT-1 pathway and cell wall integrity signaling.

• Post-translational modification profiling: Comprehensively identify all modifications on VTS1 and determine how they change in response to different environmental conditions.

• Systems biology approaches: Develop mathematical models integrating transcriptional, post-transcriptional, and post-translational regulation involving VTS1 to predict cellular responses to environmental changes.

• Comparative studies across fungal species: Compare VTS1 function across multiple fungal species to understand evolutionary conservation and divergence of RNA regulatory networks.

These research directions would not only advance our understanding of VTS1 specifically but would also contribute to broader knowledge of post-transcriptional regulation in filamentous fungi and potentially identify novel regulatory mechanisms applicable to other eukaryotes.