Recombinant Neurospora crassa Pescadillo homolog (nop-7), partial

What is Pescadillo homolog (nop-7) in Neurospora crassa?

Pescadillo homolog (nop-7) is a nucleolar protein identified in Neurospora crassa that belongs to a highly conserved family of proteins involved in ribosome biogenesis. The protein is also known as Nucleolar protein 7 homolog and has the UniProt accession number Q7SFC2. It is functionally similar to the Nop7 protein characterized in Saccharomyces cerevisiae, where it forms part of the PeBoW complex (Pes1-Bop1-WDR12 in humans) that is essential for processing of pre-rRNAs during 60S ribosomal subunit maturation .

What biological role does nop-7 play in ribosome biogenesis?

Nop7 plays a critical role in the assembly and maturation of the 60S ribosomal subunit. Studies in Saccharomyces cerevisiae have definitely associated Nop7 to ribosome biogenesis because mutations within the gene were synthetically lethal . As part of the PeBoW complex, nop-7 is required for the processing of the 27SA3 pre-rRNA to the mature 5.8S and 25S rRNAs. The protein likely functions as a structural scaffold that facilitates the association of other processing factors with pre-ribosomes.

How is the recombinant nop-7 protein typically produced?

Recombinant nop-7 is typically produced in E. coli expression systems using standardized molecular biology techniques. According to available product information, commercially available recombinant nop-7 is often partial (not full-length) and may contain various tags to aid in purification . The typical workflow involves:

• Cloning the nop-7 coding sequence into an appropriate expression vector

• Transformation of E. coli expression strains

• Induction of protein expression (often using IPTG for T7-based systems)

• Cell lysis and initial purification

• Affinity chromatography using the fusion tag

• Further purification steps such as ion exchange or size exclusion chromatography

What are the optimal storage conditions for recombinant nop-7?

For optimal stability and activity retention, recombinant nop-7 should be stored according to the following guidelines:

For working with the protein, it is recommended to:

• Briefly centrifuge the vial prior to opening

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot the protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

How does nop-7 interact with other proteins in ribosomal assembly pathways?

Nop7 functions as part of a conserved complex in the ribosome biogenesis pathway. Based on studies in yeast and other fungi, nop-7 interacts primarily with Erb1 and Ytm1 to form a stable complex that associates with pre-60S particles. The N-terminal region of Nop7 interacts with Erb1, while Ytm1 interacts with the C-terminal β-propeller domain of Erb1 .

The interaction network can be experimentally mapped using:

• Co-immunoprecipitation followed by mass spectrometry

• Yeast two-hybrid assays

• Proximity-based labeling methods (BioID or APEX)

• Structural studies using X-ray crystallography or cryo-electron microscopy

The Nop7-Erb1-Ytm1 complex appears to function in coordinating the processing and maturation of pre-60S ribosomal particles, with mutations in the interface between Erb1 and Ytm1 negatively affecting growth in yeast due to interference with 60S production .

What are the key regulatory mechanisms controlling nop-7 expression in N. crassa?

While specific information about nop-7 regulation in N. crassa is limited in the provided sources, research on related nucleolar proteins in fungi suggests multiple regulatory mechanisms:

• Transcriptional regulation: Like other genes involved in ribosome biogenesis, nop-7 expression is likely coordinated with cellular growth rates and nutritional status

• Post-translational modifications: Phosphorylation may regulate nucleolar localization and protein-protein interactions

• Protein stability control: Ubiquitin-mediated degradation pathways may regulate nop-7 protein levels

Research methodologies to study nop-7 regulation include:

• Reporter gene assays to analyze promoter activity

• ChIP-seq to identify transcription factors binding to the nop-7 promoter

• Phosphoproteomic analysis to identify regulatory phosphorylation sites

• Pulse-chase experiments to determine protein half-life

How can I design experiments to study nop-7 function using CRISPR/Cas9?

When designing CRISPR/Cas9 experiments for nop-7 functional studies in N. crassa, consider the following approach:

• Guide RNA design:

  • Select target sites with minimal off-target effects

  • Design gRNAs targeting functional domains identified through sequence alignment

  • Include controls targeting non-essential regions

• Delivery method:

  • Transformation of RNP complexes (Cas9 protein + gRNA)

  • Plasmid-based expression of Cas9 and gRNA

  • Consider using a selectable marker for screening

• Verification strategy:

  • PCR amplification and sequencing of the target region

  • Western blotting to confirm protein knockout/modification

  • Phenotypic analysis (growth rate, morphology)

• Functional analysis:

  • Ribosome profiling to assess impact on translation

  • Northern blotting to examine pre-rRNA processing

  • Polysome profiling to evaluate ribosome assembly

Remember that when working with recombinant DNA in N. crassa, experiments must comply with NIH Guidelines for Research Involving Recombinant DNA, which may require Institutional Biosafety Committee (IBC) approval before initiation .

What structural features characterize the nop-7 protein and how do they relate to function?

The structural features of nop-7 from N. crassa can be inferred from studies of homologous proteins:

• N-terminal domain: Likely contains protein-protein interaction motifs that mediate binding to Erb1

• Central region: May contain RNA-binding motifs

• C-terminal domain: Potentially involved in nucleolar localization

To experimentally determine the structure:

• X-ray crystallography of purified protein or domains

• NMR spectroscopy for smaller domains

• Cryo-EM in complex with interaction partners

• Homology modeling based on structures of homologous proteins

Research has shown that the β-propeller domain in the related protein Erb1 plays a crucial role in protein-protein interactions, specifically binding to Ytm1. This interaction is essential for 60S ribosomal subunit maturation .

How does N. crassa nop-7 compare to its homologs in other fungi and eukaryotes?

Pescadillo homologs are highly conserved across eukaryotes, suggesting an essential role in ribosome biogenesis. A comparative analysis would include:

*Estimated based on typical conservation patterns of nucleolar proteins

Methodological approaches for comparative studies:

• Multiple sequence alignment using tools like Clustal Omega or MUSCLE

• Phylogenetic analysis to trace evolutionary history

• Domain conservation analysis

• Complementation studies (can human PES1 rescue nop-7 deletion in N. crassa?)

What are the best approaches for studying nop-7 interactions with RNA?

Investigating nop-7's interactions with RNA requires specialized techniques:

• RNA immunoprecipitation (RIP):

  • Use anti-nop-7 antibodies to precipitate protein-RNA complexes

  • Analyze bound RNAs by RT-PCR or RNA-seq

• CLIP-seq (Crosslinking and Immunoprecipitation followed by sequencing):

  • UV crosslinking to capture direct RNA-protein interactions

  • Immunoprecipitation of nop-7

  • Library preparation and high-throughput sequencing

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified recombinant nop-7 with labeled RNA

  • Analyze complex formation by gel electrophoresis

• Surface Plasmon Resonance (SPR):

  • Quantitative measurement of binding kinetics

  • Determine affinity constants for different RNA substrates

• RNA structural probing in the presence/absence of nop-7:

  • SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension)

  • Hydroxyl radical footprinting

Similar approaches have been used to demonstrate that the β-propeller domain of Erb1 (a nop-7 interaction partner) binds RNA in vitro, a property that might be important for its function in ribosome biogenesis .

How do I interpret contradictory results in nop-7 functional studies?

When faced with contradictory results regarding nop-7 function, consider the following systematic approach:

• Experimental conditions analysis:

  • Compare growth conditions (media, temperature, time points)

  • Evaluate strain backgrounds (wild-type vs. laboratory strains)

  • Assess protein expression levels in different systems

• Technical variables:

  • Antibody specificity and validation

  • Purification methods and protein folding

  • Tag interference with protein function

• Biological complexity considerations:

  • Redundant pathways that may compensate for nop-7 disruption

  • Conditional phenotypes that appear only under specific stress conditions

  • Interactions with other cellular processes

• Resolution strategies:

  • Use multiple complementary techniques to address the same question

  • Perform rescue experiments with wild-type protein

  • Design domain-specific mutations to dissect function

  • Collaborate with groups reporting contradictory results

When interpreting results, remember that extensive analysis of Δnop-7 strains in N. crassa may not reveal obvious defects under normal laboratory conditions, similar to findings with other nucleolar proteins like nop-1 .

What quality control measures should be implemented when working with recombinant nop-7?

Ensuring the quality and consistency of recombinant nop-7 preparations is critical for reliable research outcomes:

• Protein purity assessment:

  • SDS-PAGE with Coomassie or silver staining (expected purity >85%)

  • Mass spectrometry for identity confirmation

  • Size exclusion chromatography to detect aggregation

• Functional validation:

  • RNA binding assays

  • Interaction studies with known binding partners (e.g., Erb1)

  • Circular dichroism to assess secondary structure

• Storage stability monitoring:

  • Regular activity tests after storage at different temperatures

  • Freeze-thaw stability assessment

  • Comparison of fresh vs. stored protein activity

• Batch-to-batch consistency:

  • Standard operating procedures for expression and purification

  • Reference standards for comparison

  • Detailed record-keeping of expression conditions

For protein reconstitution, it's recommended to reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol (final concentration) added for stability .

How can nop-7 research contribute to our understanding of eukaryotic ribosome biogenesis?

Research on nop-7 in N. crassa provides several advantages for understanding fundamental aspects of eukaryotic ribosome biogenesis:

• Evolutionary insights:

  • N. crassa represents a filamentous fungal model system with aspects of both unicellular and multicellular biology

  • Comparative studies can reveal conserved and divergent mechanisms across eukaryotes

• Technical advantages:

  • Genetic tractability of N. crassa

  • Well-characterized genome (chromosome VII has been extensively studied)

  • Ability to perform large-scale screens

• Specific contributions to ribosome biogenesis understanding:

  • Elucidation of assembly factor networks

  • Identification of regulatory checkpoints

  • Understanding of quality control mechanisms

• Translational relevance:

  • Ribosomopathies in humans are linked to mutations in ribosome biogenesis factors

  • Cancer cells often show dysregulation of ribosome production

  • Potential for identifying novel antifungal targets

Methodological approaches combining genetics, biochemistry, and structural biology have proven particularly powerful in this field, as demonstrated by studies of the Nop7-Erb1-Ytm1 complex that revealed important insights into 60S ribosomal subunit assembly .