Recombinant Neurospora crassa 40S ribosomal protein S25 (rps-25)

What is the structural characterization of Neurospora crassa rps-25 and how does it compare to homologs in other eukaryotes?

Neurospora crassa rps-25 is a small ribosomal protein located in the head domain of the 40S ribosomal subunit. While specific structural data for N. crassa rps-25 is limited, comparative analysis with homologs provides valuable insights. For example, rat ribosomal protein S25 consists of 125 amino acids with a molecular weight of 13,733 daltons . RPS25 is positioned in the E site of the 40S subunit, contacting helix 41 of the 18S rRNA and situated between ribosomal proteins S5 and S18 .

Based on evolutionary conservation patterns, N. crassa rps-25 likely shares significant structural similarities with yeast and mammalian homologs. The protein is expected to have conserved domains that facilitate interactions with rRNA and other ribosomal components, particularly in regions critical for specialized translation functions. Sequence alignment and structural prediction methodologies are essential for characterizing these features in the absence of crystal structure data.

What techniques are recommended for purifying recombinant N. crassa rps-25 while maintaining its native conformation and function?

Purification of functional recombinant N. crassa rps-25 requires careful consideration of expression systems and purification strategies. A recommended methodological approach includes:

• Expression system selection: E. coli BL21(DE3) strains with codon optimization for N. crassa preferred codons yield higher expression levels. Alternatively, yeast expression systems may preserve eukaryotic post-translational modifications.

• Construct design: Incorporate a cleavable His-tag or GST-tag to facilitate purification while allowing tag removal to study native protein function.

• Solubility enhancement: Express at lower temperatures (16-18°C) with reduced IPTG concentration (0.1-0.5 mM) to improve proper folding.

• Purification protocol:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

  • Intermediate purification via ion exchange chromatography

  • Polishing step using size exclusion chromatography to ensure homogeneity

• Functional validation: Ribosome binding assays and in vitro translation systems to confirm activity preservation.

Special consideration should be given to buffer composition, including the presence of reducing agents to maintain cysteine residues in their reduced state and low concentrations of nuclease inhibitors to prevent RNA degradation from contaminating nucleases.

How conserved is rps-25 across fungal species, and what can phylogenetic analysis reveal about its evolution and specialized functions?

Ribosomal protein S25 demonstrates significant conservation across eukaryotic lineages, suggesting fundamental roles in translation. Comparative analysis reveals that N. crassa ribosomal proteins share homology with counterparts in both yeast and humans. For example, another N. crassa ribosomal protein, crp-3, shares 89% sequence homology at its N-terminus with yeast rp51 and 83% with human S17 .

For rps-25 specifically, the conservation pattern is likely similar, with highest sequence identity in functional domains that interact with rRNA and other ribosomal components. Phylogenetic analysis methodology should include:

• Multiple sequence alignment of rps-25 sequences from diverse fungal species, including ascomycetes, basidiomycetes, and early-diverging fungi.

• Identification of conserved motifs using tools like MEME and GLAM2.

• Calculation of selection pressures (dN/dS ratios) to identify regions under purifying or positive selection.

• Ancestral sequence reconstruction to trace the evolutionary history of key functional domains.

Interestingly, despite sequence conservation, intron positions are often not conserved between homologous genes across species. For instance, the crp-3 gene in N. crassa contains two introns that are not positionally conserved with its yeast and human homologs , suggesting independent evolutionary events affecting gene structure even while protein sequence remains conserved.

What genomic features characterize the rps-25 gene locus in Neurospora crassa, and how do regulatory elements compare to other ribosomal protein genes?

The genomic organization of ribosomal protein genes in N. crassa reveals coordinated regulatory mechanisms. While specific information about the rps-25 locus is limited in the provided search results, insights can be drawn from studies of other N. crassa ribosomal protein genes. Ribosomal protein genes in N. crassa share several common upstream regulatory elements, including:

• A 'Taq box' with consensus sequence ARTTYGACTT located approximately 39 bp upstream of the transcription start site.

• A CG repeat region with consensus sequence CCCRCCRRR at around position -65.

• A major transcription initiation site embedded in a purine-rich region flanked by an upstream pyrimidine-rich sequence .

These elements likely play crucial roles in the coordinated expression of ribosomal protein genes, including rps-25. The rps-25 promoter would be expected to contain these regulatory elements to ensure synchronized expression with other ribosomal components.

For comprehensive genomic characterization of the rps-25 locus, researchers should employ:

• Promoter analysis using reporter gene assays

• ChIP-seq to identify transcription factor binding sites

• Comparative genomics to identify conserved non-coding regulatory elements

• CRISPR-mediated genome editing to validate functional elements

What role does rps-25 play in alternative translation initiation mechanisms in Neurospora crassa?

While specific data for N. crassa rps-25 is limited in the search results, studies in other eukaryotic systems reveal that RPS25 plays critical roles in alternative translation initiation mechanisms without affecting canonical cap-dependent translation. Based on these findings, N. crassa rps-25 likely exhibits similar specialized functions.

RPS25 is specifically required for at least two alternative translation pathways:

• Internal Ribosome Entry Site (IRES)-mediated translation: RPS25 is essential for efficient translation initiation via both hepatitis C virus (HCV)-like and intergenic region (IGR)-like IRES elements .

• Ribosome shunting: This mechanism involves ribosome recruitment to the mRNA 5' end through cap-dependent means followed by scanning and "shunting" across structured regions. RPS25 is required for this process as well .

The dependency on RPS25 for these alternative mechanisms suggests a common feature in their initiation pathways that differs from cap-dependent translation. To investigate this in N. crassa specifically, researchers should:

• Generate RPS25-depleted N. crassa strains through CRISPR-Cas9 or RNAi approaches

• Assess translation efficiency of reporter constructs containing IRES elements or shunting-dependent structures

• Analyze polysome profiles to determine effects on global translation

• Employ ribosome profiling to identify mRNAs with altered translation efficiency in the absence of rps-25

How does rps-25 contribute to stress response translation in Neurospora crassa?

During cellular stress conditions, cap-dependent translation is typically downregulated while alternative translation mechanisms are employed to synthesize stress-response proteins. Given RPS25's established role in alternative translation initiation, it likely plays a significant role in stress response translation in N. crassa.

Ribosomal proteins in N. crassa, including likely rps-25, show coordinated regulation during nutritional stress. For example, when N. crassa undergoes a nutritional downshift from sucrose to quinic acid, the levels of ribosomal protein mRNAs are closely coordinated , indicating a synchronized response to stress conditions.

To investigate rps-25's role in stress response translation in N. crassa, recommended methodological approaches include:

• Comparative transcriptomics and proteomics of wild-type vs. rps-25 knockout/knockdown strains under various stress conditions (nutrient limitation, oxidative stress, heat shock)

• Polysome profiling coupled with RNA-seq to identify stress-responsive mRNAs that show altered translation efficiency in the absence of rps-25

• Reporter assays using stress-responsive gene 5'UTRs fused to luciferase to assess translation efficiency

• RNA structure probing of stress-responsive mRNAs to identify potential structural elements that might require rps-25 for efficient translation

The data would likely reveal a subset of stress-responsive genes that rely heavily on rps-25 for their translation, potentially identifying a stress-specific translational program in N. crassa.

What are the optimal conditions for expressing recombinant N. crassa rps-25 for structural studies?

For successful structural studies of recombinant N. crassa rps-25, optimized expression conditions are critical. Based on properties of ribosomal proteins and their expression challenges, the following methodological approach is recommended:

Expression system selection:

• Bacterial expression: E. coli BL21(DE3) Rosetta strain is preferred for addressing codon bias issues

• Yeast expression: Pichia pastoris for maintaining post-translational modifications

• Insect cell expression: Baculovirus system for complex eukaryotic proteins

Optimization parameters:

Solubility enhancement strategies:

• Fusion tags: Thioredoxin (TRX) or SUMO tags significantly improve solubility

• Co-expression with chaperones: GroEL/GroES system

• Buffer optimization: Include 5% glycerol, 1mM DTT, and low concentrations of arginine (50-100mM)

For structural studies specifically, uniformly 15N/13C-labeled protein can be produced using M9 minimal media with 15NH4Cl and 13C-glucose for NMR studies, or selenomethionine incorporation for X-ray crystallography phasing.

What strategies are most effective for characterizing protein-protein interactions involving rps-25 within the Neurospora crassa ribosome?

Characterizing protein-protein interactions of rps-25 within the N. crassa ribosome requires specialized approaches that preserve native ribosomal architecture. The following methodological strategies are recommended:

In vivo approaches:

• Proximity-dependent labeling methods:

  • BioID or TurboID fused to rps-25

  • APEX2 proximity labeling
    These methods identify proteins in close proximity to rps-25 under native conditions

• Fluorescence microscopy techniques:

  • FRET pairs between rps-25 and suspected interaction partners

  • Split fluorescent protein complementation assays

In vitro approaches:

• Crosslinking mass spectrometry (XL-MS):

  • Chemical crosslinkers (DSS, BS3) followed by mass spectrometry

  • UV-crosslinking for RNA-protein interactions

• Cryo-electron microscopy:

  • Structure determination of intact N. crassa ribosomes

  • Comparison with ribosomes lacking rps-25

Interaction validation:

• Co-immunoprecipitation with tagged rps-25

• Surface plasmon resonance (SPR) with purified components

• Microscale thermophoresis (MST) for interaction affinity measurement

For functional validation, researcher should employ in vitro translation assays using IRES-containing reporter constructs with purified ribosomes from wild-type and rps-25 knockout strains to assess the impact of specific interactions on alternative translation mechanisms.

How is the expression of rps-25 coordinated with other ribosomal proteins in Neurospora crassa during different growth conditions?

Ribosomal protein genes in N. crassa display coordinated regulation in response to growth conditions and carbon source availability. Studies indicate that rRNA and ribosomal protein levels are coordinately regulated by growth rate and carbon nutrition . While specific data on rps-25 regulation is not provided in the search results, insights from other ribosomal protein genes suggest similar regulatory patterns.

The expression of N. crassa ribosomal protein genes is closely coordinated during nutritional shifts. For example, when N. crassa undergoes a nutritional downshift from sucrose to quinic acid, the levels of ribosomal protein mRNAs change in a synchronized manner . This coordination suggests common regulatory mechanisms governing all ribosomal protein genes, likely including rps-25.

Methodological approaches to investigate rps-25 coordinated expression include:

• Time-course RNA-seq analysis during nutritional shifts, comparing rps-25 expression patterns with other ribosomal protein genes

• ChIP-seq experiments to identify transcription factors binding to the rps-25 promoter under different conditions

• Promoter analysis to identify regulatory elements shared with other ribosomal protein genes, particularly the 'Taq box' (consensus: ARTTYGACTT) and CG repeat (consensus: CCCRCCRRR) elements found in other N. crassa ribosomal protein genes

• Reporter assays with full and truncated rps-25 promoter constructs to determine the functional significance of identified regulatory elements

• Metabolic labeling experiments to assess protein synthesis rates and stability under different growth conditions

What role does rps-25 play in the response of Neurospora crassa to nutrient limitation and other stress conditions?

Ribosomal proteins in N. crassa, likely including rps-25, play important roles in adapting translation to stress conditions. Based on the search results and broader understanding of ribosomal protein function, rps-25 may be particularly important during stress due to its role in alternative translation mechanisms.

The relative levels of rRNAs and ribosomal proteins in N. crassa are coordinately regulated by growth rate and carbon nutrition , suggesting that nutrient limitation triggers a specific ribosomal protein expression program. Given RPS25's specialized role in alternative translation mechanisms like IRES-mediated translation and ribosome shunting , it may be particularly important during stress conditions when cap-dependent translation is inhibited.

To investigate rps-25's specific role in stress response, researchers should employ:

• Comparative growth assays of wild-type vs. rps-25 knockout/knockdown strains under various stress conditions:

  • Carbon source limitation

  • Nitrogen source limitation

  • Oxidative stress (H₂O₂ exposure)

  • Heat shock

  • Osmotic stress

• Transcriptome and translatome analysis (using techniques like ribosome profiling) to identify mRNAs differentially translated in the absence of rps-25 during stress

• Reporter assays using stress-responsive gene 5'UTRs to determine if they contain IRES-like elements or other features that might confer rps-25 dependency

• Metabolic flux analysis to determine if rps-25 influences adaptations in central carbon metabolism during nutrient limitation

Could N. crassa rps-25 knockouts serve as a model for studying translation-related neurodegenerative diseases?

The search results reveal that RPS25 plays a critical role in RAN (repeat-associated non-AUG) translation of nucleotide repeat expansions associated with neurodegenerative diseases such as ALS and FTD . This suggests that N. crassa rps-25 knockouts could indeed serve as valuable models for studying translation mechanisms involved in neurodegenerative disorders.

RPS25 knockout has been shown to reduce the translation of repeats associated with C9orf72 ALS/FTD, as well as CAG repeats in ATXN2 and HTT genes . In human cells, RPS25 knockout reduced poly(GP) RAN products by ~50% and poly(GA) products by over 90% . This effect was specific to RAN translation and did not significantly impact global translation or cell growth rates.

To develop N. crassa as a model system for studying these mechanisms, researchers should:

• Generate rps-25 knockout strains in N. crassa using CRISPR-Cas9 or traditional knockout methods

• Create expression constructs containing disease-associated repeat sequences (GGGGCC, CAG) in N. crassa

• Assess RAN translation of these repeats in wild-type vs. rps-25 knockout strains

• Compare polysome profiles and conduct ribosome profiling to determine effects on global translation

• Perform structural studies comparing wild-type and rps-25-deficient ribosomes to identify conformational changes that might explain RAN translation dependence

This model system would provide several advantages:

• Faster growth than mammalian models

• Sophisticated genetic tools available in N. crassa

• Ability to perform high-throughput screens for modifiers of RAN translation

• Evolutionary insights through comparison with mammalian systems

How can structural data from N. crassa rps-25 inform the development of selective translation inhibitors for therapeutic applications?

Structural characterization of N. crassa rps-25 could significantly contribute to rational drug design efforts targeting selective translation mechanisms. Given RPS25's role in alternative translation initiation without affecting global cap-dependent translation , it presents a unique target for developing selective inhibitors.

The search results indicate that RPS25 is located in the E site of the ribosome, contacting helix 41 of the 18S rRNA and positioned between ribosomal proteins S5 and S18 . Structural studies of N. crassa rps-25 could reveal fungal-specific features that differ from human RPS25, potentially enabling the development of selective antifungal compounds.

Additionally, RPS25's essential role in RAN translation of disease-associated repeat expansions suggests that compounds targeting human RPS25-specific functions could have therapeutic potential for neurodegenerative diseases like ALS/FTD, spinocerebellar ataxias, and Huntington's disease.

Methodological approach for structure-based drug discovery:

• High-resolution structural determination:

  • Cryo-EM of N. crassa ribosomes with and without rps-25

  • X-ray crystallography of isolated rps-25 alone or in complex with interacting partners

  • NMR studies of dynamic regions

• Identification of druggable pockets:

  • Computational analysis of potential binding sites

  • Comparison with human RPS25 to identify unique structural features

  • Molecular dynamics simulations to reveal transient pockets

• Virtual screening and fragment-based approaches:

  • In silico screening against identified pockets

  • Fragment screening using NMR or thermal shift assays

  • Structure-activity relationship studies

• Functional validation:

  • In vitro translation assays using IRES or RAN reporters

  • Cell-based assays in disease models

  • Selectivity profiling against cap-dependent translation

How can CRISPR-Cas9 genome editing be optimized for studying rps-25 function in Neurospora crassa?

CRISPR-Cas9 genome editing in N. crassa requires specific optimizations to achieve efficient targeting of ribosomal protein genes like rps-25, which may be essential or present in multiple copies. Based on the search results indicating that rat S25 may have 19-22 gene copies , genomic analysis of N. crassa should first confirm the copy number of rps-25.

Optimized CRISPR-Cas9 methodology for N. crassa rps-25 functional studies:

Construct design considerations:

• Selection of appropriate promoters:

  • trpC promoter for Cas9 expression

  • U6 snRNA promoter for sgRNA expression

• sgRNA design specifics:

  • Target unique regions of rps-25 to avoid off-target effects

  • Design multiple sgRNAs targeting different exons

  • Validate sgRNA efficiency using in silico tools optimized for N. crassa genome

Delivery methods:

• Polyethylene glycol (PEG)-mediated transformation of germinated conidia

• Agrobacterium tumefaciens-mediated transformation for complex constructs

• Biolistic delivery for difficult-to-transform strains

Genetic modification strategies:

• Complete knockout (if non-essential):

  • Design repair templates with selectable markers

  • Screen for homology-directed repair events

• Conditional depletion (if essential):

  • Implement a tetracycline-repressible promoter system

  • Create an auxin-inducible degron tag fusion

• Point mutations to study specific functions:

  • Design ssODN repair templates with desired mutations

  • Include silent mutations to create restriction sites for screening

• Tagging for localization and interaction studies:

  • C-terminal GFP or split fluorescent protein tags

  • Affinity tags (FLAG, HA) for purification and interaction studies

Validation approaches:

• Sanger sequencing of genomic locus

• Western blotting to confirm protein expression changes

• Ribosome profiling to assess functional impacts

• Growth rate and morphology analysis under various conditions

What approaches can reveal the role of rps-25 in regulating selective translation of specific mRNAs in Neurospora crassa?

Investigating rps-25's role in selective mRNA translation requires comprehensive translatomic approaches. Given RPS25's established role in alternative translation mechanisms like IRES-mediated translation and ribosome shunting , it likely influences the translation of specific mRNA subsets in N. crassa.

Methodological approaches to identify and characterize these mRNAs include:

Genome-wide translational profiling:

• Ribosome profiling (Ribo-seq) comparing wild-type and rps-25 knockout/knockdown strains:

  • Under normal growth conditions

  • During various stress conditions (nutrient limitation, heat shock)

  • During different developmental stages

• Polysome profiling coupled with RNA-seq to identify mRNAs with altered ribosome association in the absence of rps-25

• Translating ribosome affinity purification (TRAP) to isolate mRNAs associated with tagged ribosomes in specific cellular compartments

mRNA structural analysis:

• SHAPE-seq or DMS-seq to identify structured regions in 5'UTRs that might function as IRES elements

• Comparative analysis of 5'UTR features in mRNAs dependent on rps-25 for translation

• In vitro structure probing of candidate IRES elements

Functional validation:

• Reporter assays using 5'UTRs from candidate mRNAs fused to luciferase

• CRISPR-mediated mutagenesis of identified structural elements

• In vitro translation assays with purified components

• Toeprinting assays to map precise ribosome positioning on candidate mRNAs

Proteomics approaches:

• Stable isotope labeling (SILAC) to quantify newly synthesized proteins

• Pulse-chase experiments to determine protein synthesis rates

• Global proteome analysis comparing wild-type and rps-25 mutant strains

The results would likely reveal classes of mRNAs particularly dependent on rps-25 for translation, potentially identifying IRES-like elements in fungal mRNAs and expanding our understanding of specialized translation mechanisms in N. crassa.