Recombinant Neurospora crassa 60S ribosomal protein L24 (rpl-24)

What is the genomic organization of the rpl-24 gene in Neurospora crassa?

The rpl-24 gene in Neurospora crassa (NCU03150) encodes the 60S ribosomal protein L24, a component of the large ribosomal subunit. Unlike many yeast ribosomal protein genes, N. crassa ribosomal protein genes typically contain multiple introns. Comparative analysis with other eukaryotic ribosomal proteins shows that the coding region is often interrupted by several intervening sequences, similar to the pattern observed in the N. crassa gene homologous to the yeast CYH2 gene, which contains seven intervening sequences . The genomic sequence is available in public databases with the accession numbers including XM_959107.3 .

How conserved is RPL24 across fungal species compared to other ribosomal proteins?

RPL24 shows significant conservation across fungal species, particularly within its functional domains. Comparative analyses reveal:

This level of conservation suggests RPL24 has an important role in ribosomal function that has been maintained throughout evolution. The pattern of codon usage in the N. crassa rpl-24 gene is highly biased, consistent with high translation efficiency, which is typical of ribosomal protein genes . Unlike some ribosomal proteins that have undergone duplication events, rpl-24 is present as a single copy in the N. crassa genome, mapped to a specific chromosomal locus .

What is the optimal expression system for producing recombinant N. crassa RPL24 protein?

Based on existing research, E. coli expression systems have proven effective for the production of recombinant N. crassa RPL24. The most successful approach involves:

• Cloning the full-length coding sequence (amino acids 1-310) into an expression vector with an N-terminal His-tag

• Expression in E. coli under T7 promoter control

• Induction with IPTG at lower temperatures (18-25°C) to enhance proper folding

• Purification using immobilized metal affinity chromatography

The resulting protein can be obtained with >90% purity as determined by SDS-PAGE. For long-term storage, lyophilization of the protein in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 is recommended . For reconstitution, deionized sterile water should be used to reach a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

How can I efficiently create targeted mutations in the rpl-24 gene of Neurospora crassa?

Multiple approaches are available for targeted mutagenesis of the rpl-24 gene in N. crassa, with CRISPR/Cas9 being the most efficient recent method:

A. CRISPR/Cas9 System:

• Incorporate the cas9 sequence into the fungal genome using a selectable marker

• Design guide RNAs (gRNAs) targeting specific regions of the rpl-24 gene

• Introduce naked gRNA via electroporation

• Use cyclosporin-resistant-1 (csr-1) as a selectable marker gene for identification of transformants

This approach has demonstrated 100% editing efficiency under selection conditions in N. crassa . It eliminates the need for constructing multiple vectors, significantly accelerating the mutagenesis process.

B. Sequential Repetitive Gene Deletions:
For sequential modifications, a bacterial recombination system employing β-recombinase acting on six recognition sequences (β-rec/six) allows repetitive site-specific gene deletion and marker recycling in N. crassa .

How does cycloheximide (CHX) interact with RPL24 in Neurospora crassa ribosomes?

Cycloheximide (CHX) arrests the elongation function of most eukaryotic ribosomes by binding near the E site of the large subunit (LSU). In N. crassa, high-resolution cryo-electron microscopy (cryo-EM) studies at 2.7 Å resolution have revealed:

• CHX binding position is highly conserved between N. crassa, S. cerevisiae, and human ribosomes

• The binding pocket is formed by eL42, conserved 26S rRNA residues, and uL15

• Unlike canonical CHX-bound structures in yeast and human ribosomes which contain a Mg²⁺ ion adjacent to the CHX-binding pocket, N. crassa ribosomes contain spermidine (SPD) in this position

Importantly, mutations conferring CHX resistance in N. crassa map to conserved residues in the CHX-binding pocket. CHX does not appear to interfere with termination as it does with elongation, consistent with CHX interfering with the translocation of tRNA to the E site but not peptidyl transfer events at the A site .

What is the impact of RPL24 depletion on translation elongation rates in eukaryotic cells?

Studies on RPL24 depletion, primarily from mouse models with the Rpl24Bst mutation, demonstrate significant effects on translation elongation:

Interestingly, RPL24 depletion does not alter the available pool of ribosomal subunits as previously suggested, but instead alters signaling that regulates translation factors . These findings position translation elongation as a potential therapeutic target in certain cancers where RPL24 depletion has shown tumor-suppressive effects .

How can N. crassa RPL24 be used in systematic reviews of translation control mechanisms?

N. crassa RPL24 serves as an excellent model for systematic reviews of translation control due to several advantages:

• Methodological Approach:

  • Define precise research questions about RPL24's role in translation control

  • Search multiple databases (MEDLINE, Embase, Web of Science) using standardized terms

  • Apply rigorous inclusion/exclusion criteria to identify high-quality studies

  • Extract data on RPL24 function across multiple organisms for comparative analysis

  • Assess study quality using tools like AMSTAR 2

• Key Parameters to Analyze:

  • Structural conservation of RPL24 across species

  • Mutational effects on ribosome assembly and function

  • Context-dependent roles in different tissues or developmental stages

  • Interactions with other translation factors

  • Post-translational modifications affecting function

Such systematic reviews should follow established protocols and include PRISMA diagrams to document the search and selection process . Data synthesis should involve tabulation of key findings with careful attention to methodological heterogeneity across studies.

How does the structure of N. crassa ribosomes containing RPL24 differ from those of other model organisms?

Structural analysis of N. crassa ribosomes reveals distinct features compared to other model organisms:

This structural information provides important insights into the evolution of ribosome structure and function across eukaryotes.

What are the most effective strategies for studying rpl-24 function through genetic manipulation in N. crassa?

Several complementary approaches can be employed to study rpl-24 function in N. crassa:

A. Regulated Expression Systems:

• Replace the native rpl-24 promoter with an inducible promoter (e.g., qa-2) using homologous recombination

• Create conditional knockdown strains using RNAi or antisense expression

• Employ the cre-1 regulated carbon-source dependent expression system

B. Mutation Analysis:

• Introduce specific point mutations based on structural data to target functional domains

• Create hypomorphic alleles that partially reduce function

• Use the CRISPR/Cas9 system with repair templates containing desired mutations

C. Reporter Systems:

• Tag RPL24 with fluorescent proteins to track localization and dynamics

• Use pull-down assays to identify interaction partners

• Employ ribosome profiling to assess global translation effects

When designing these experiments, it's essential to use factorial experimental designs that consider multiple variables simultaneously. For example, a 2³ factorial design could examine the effects of temperature, carbon source, and strain background on RPL24 function .

How can I design experiments to study the effect of rpl-24 mutations on stress response in N. crassa?

A comprehensive experimental design to study rpl-24 mutations and stress response should include:

• Strain Generation:

  • Create precise mutations in rpl-24 using CRISPR/Cas9

  • Generate a series of mutants with varying degrees of functional impairment

  • Include appropriate control strains (wild-type, complemented mutants)

• Experimental Variables:

  • Stress conditions: heat shock, oxidative stress, osmotic stress, nutrient limitation

  • Growth phases: germination, exponential growth, stationary phase

  • Media compositions: minimal vs. complete, different carbon sources

• Experimental Design Matrix:

• Analytical Methods:

  • Growth rate determination

  • Polysome profiling to assess translation status

  • ³⁵S-methionine incorporation to measure protein synthesis

  • RNA-seq to identify differentially expressed genes

  • Western blotting for stress-specific markers

  • Cellular imaging for morphological changes

This design incorporates multiple factors and response variables, allowing for the detection of interaction effects between rpl-24 mutations and specific stress conditions .

How does the function of RPL24 in N. crassa compare to its role in cancer suppression observed in mouse models?

Studies comparing RPL24 function across species reveal important similarities and differences:

Similarities:

• In both N. crassa and mouse models, RPL24 is critical for efficient translation

• Reduced RPL24 expression affects translation elongation in both systems

• RPL24 impacts phosphorylation of eEF2 (eukaryotic elongation factor 2) across species

Key Differences:

• In mouse models (Rpl24Bst), RPL24 depletion suppresses colorectal cancer by promoting eEF2 phosphorylation, but this cancer-specific effect has not been studied in N. crassa

• Mouse Rpl24Bst mutations suppress translation and limit tumorigenesis in models with Apc deletion and Kras mutations

• The suppressive effect is specific to Kras-mutant cells and does not occur in Kras wild-type models

• RPL24 depletion in mouse models does not alter ribosomal subunit abundance but specifically affects translation elongation through eEF2 phosphorylation

This cross-species comparison suggests evolutionarily conserved mechanisms of translation control that have been adapted for different functions in complex multicellular organisms, such as tumor suppression in mammals.

What are the structural and functional variations in RPL24 across different fungal species?

Analysis of RPL24 across fungal species reveals both conservation and divergence:

• Sequence Conservation:

  • Core functional domains show high conservation (70-80% identity)

  • N- and C-terminal regions display greater variability

  • RNA binding motifs are highly conserved

• Structural Features:

  • Secondary structure elements are preserved across species

  • Surface-exposed loops show greater variability

  • Interaction interfaces with other ribosomal components are highly conserved

• Functional Implications:

  • Role in ribosome assembly appears universally conserved

  • Response to translation inhibitors (e.g., cycloheximide) shows species-specific variations

  • Regulatory mechanisms controlling RPL24 expression differ between species

  • Some species contain multiple RPL24 paralogs, while N. crassa contains a single copy

These variations provide insights into the evolution of translation machinery across fungal lineages and can inform the development of species-specific translation inhibitors for antifungal applications.

How does RPL24 function interact with N. crassa's circadian rhythm and light response pathways?

The interaction between RPL24 and circadian/light response pathways in N. crassa involves several interconnected mechanisms:

• Translational Regulation:

  • RPL24 influences the translation efficiency of key circadian clock components

  • Under different light conditions, changes in RPL24-dependent translation affect the balance of clock proteins

  • Translation of photoreceptor proteins may be selectively regulated by RPL24-containing ribosomes

• Experimental Approaches:

  • Track luminescence reporters of clock genes in wild-type vs. rpl-24 mutant backgrounds

  • Perform ribosome profiling under different light conditions to identify differentially translated mRNAs

  • Analyze polysome distribution of circadian transcripts in rpl-24 mutants

  • Measure phase shifts and period length in circadian rhythms when RPL24 levels are altered

• Methodological Considerations:

  • Control for indirect effects of RPL24 mutation on general translation

  • Use temperature-sensitive alleles for temporal control of RPL24 function

  • Combine with mutations in known photoreceptors and clock components to assess genetic interactions

This research area connects translational control mechanisms to environmental signaling pathways, providing insights into how protein synthesis is coordinated with environmental cues .

What role does RPL24 play in the amino acid metabolism and nutrient sensing pathways of N. crassa?

RPL24's involvement in amino acid metabolism and nutrient sensing in N. crassa is multifaceted:

• Amino Acid Biosynthesis:

  • N. crassa can normally synthesize all 20 amino acids

  • RPL24-dependent translation may preferentially affect enzymes involved in amino acid biosynthesis

  • Under amino acid limitation, RPL24 function becomes particularly important for selective translation

• Experimental Evidence:

  • Growth experiments with rpl-24 mutants show differential sensitivities to amino acid availability

  • Specific amino acid auxotrophies may emerge in rpl-24 mutants under certain conditions

  • Metabolic flux analysis reveals altered amino acid biosynthesis pathways in rpl-24 mutants

• Integration with Signaling Pathways:

  • RPL24 function intersects with TOR (Target of Rapamycin) signaling

  • Amino acid starvation responses are modulated by RPL24-dependent translation

  • Cross-pathway control of amino acid biosynthesis involves translational regulation

When a Neurospora strain shows amino acid requirements (such as leucine auxotrophy), it typically indicates a mutation affecting the biochemical pathway leading to the synthesis of that specific amino acid . RPL24 mutations could potentially affect translation of enzymes in these pathways, creating similar phenotypes through a different mechanism.