Recombinant Takifugu rubripes 40S ribosomal protein S24 (rps24)

What is the basic structure of Takifugu rubripes 40S ribosomal protein S24 and how does it compare to homologs in other species?

The 40S ribosomal protein S24 (rps24) in Takifugu rubripes is a small but essential component of the 40S ribosomal subunit. While specific structural data for T. rubripes rps24 is limited, comparative analysis with other vertebrate S24 proteins suggests a conserved tertiary structure with key RNA-binding domains. Like other ribosomal proteins, rps24 contains several conserved motifs that facilitate interactions with ribosomal RNA and other ribosomal proteins.

The molecular weight of T. rubripes rps24 is estimated at approximately 15-16 kDa, consistent with ribosomal protein S24 in other vertebrates. Sequence alignment studies demonstrate high conservation among fish species, particularly within the RNA-binding domain and protein-protein interaction regions. When comparing T. rubripes rps24 with other Takifugu species like T. pseudommus and T. chinensis, the genetic similarity is extremely high due to their close evolutionary relationship .

How does the 40S ribosomal protein S24 contribute to ribosome function and regulation in T. rubripes?

The 40S ribosomal protein S24 plays several crucial roles in ribosome function:

• Structural integrity: rps24 contributes to the proper assembly and maintenance of the 40S ribosomal subunit structure.

• Translation initiation: It participates in the recruitment and positioning of mRNA during translation initiation.

• Quality control: rps24 appears to be involved in ribosome quality control pathways, particularly in the initiation RQC (iRQC) that monitors 40S ribosome integrity .

Research on ribosomal quality control pathways suggests that ubiquitylation of 40S ribosomal proteins (including potential modification of rps24) may serve as a signal for selective degradation of improperly assembled or damaged 40S subunits . This quality control mechanism ensures cellular ribosomal homeostasis by maintaining proper ratios of 40S to 60S subunits.

Experimental evidence from studies on related ribosomal proteins indicates that rps24 may participate in the recognition of specific mRNA features, potentially influencing translation selectivity in response to cellular conditions.

What post-translational modifications affect rps24 function in T. rubripes?

Post-translational modifications (PTMs) of 40S ribosomal proteins, including potential modifications of rps24, are critical regulators of ribosome function. While T. rubripes rps24-specific modifications have not been extensively characterized, research on 40S ribosomal proteins more broadly indicates several important PTMs:

• Ubiquitylation: Research on 40S ribosomal proteins has shown that ubiquitylation can target specific ribosomal proteins for degradation. This appears to be a key mechanism in ribosomal quality control .

• Phosphorylation: Phosphorylation of ribosomal proteins can modulate ribosome assembly, translation efficiency, and interactions with regulatory factors.

• Methylation and acetylation: These modifications can affect protein-RNA and protein-protein interactions within the ribosome.

Studies have demonstrated that the E3 ubiquitin ligase RNF10 can target specific 40S ribosomal proteins (particularly uS3 and uS5) for ubiquitylation, leading to reduced 40S abundance . While rps24-specific ubiquitylation has not been directly documented in the provided research, similar regulatory mechanisms may apply.

The deubiquitylating enzyme USP10 counteracts RNF10 activity, and loss of USP10 function results in increased 40S degradation . This suggests a dynamic equilibrium between ubiquitylation and deubiquitylation that regulates 40S ribosomal protein levels, potentially including rps24.

What are the optimal expression systems and conditions for producing recombinant T. rubripes rps24?

When expressing recombinant T. rubripes rps24, several expression systems and conditions should be considered:

Table 1: Recommended Expression Systems for Recombinant T. rubripes rps24

For bacterial expression systems, optimization of the following parameters is recommended:

• Growth temperature: Lower temperatures (16-18°C) after induction often improve protein solubility.

• Induction timing: Induction at mid-log phase (OD₆₀₀ of 0.6-0.8) typically yields better results.

• Expression tags: An N-terminal 6×His tag with a TEV protease cleavage site facilitates purification while allowing tag removal.

• Codon optimization: Adapting the T. rubripes rps24 sequence to E. coli codon usage improves expression efficiency.

When designing expression constructs, it's important to consider potential post-translational modifications in the native protein. If these modifications are critical for your research, a eukaryotic expression system may be preferable despite lower yields.

What purification strategies yield the highest purity and activity for recombinant rps24?

A multi-step purification approach is recommended for obtaining high-purity, functional recombinant rps24:

Step 1: Initial capture by affinity chromatography

• For His-tagged rps24: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Buffer conditions: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM imidazole (binding buffer)

• Elution with 250 mM imidazole gradient

Step 2: Intermediate purification by ion exchange chromatography

• Cation exchange chromatography (SP Sepharose) at pH 6.5-7.0

• Buffer conditions: 20 mM HEPES pH 7.0, 50 mM NaCl (binding)

• Elution with NaCl gradient (50-500 mM)

Step 3: Polishing by size exclusion chromatography

• Superdex 75 or equivalent

• Buffer conditions: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT

Throughout purification, incorporating these critical elements will help maintain protein stability:

• Include protease inhibitors in lysis buffers

• Maintain samples at 4°C

• Add reducing agents (1-5 mM DTT or 2-5 mM β-mercaptoethanol)

• Consider adding 5-10% glycerol for long-term storage

For functional studies, it's essential to verify that the recombinant protein retains its RNA-binding capability. This can be assessed through electrophoretic mobility shift assays (EMSA) using synthetic RNA oligonucleotides corresponding to known ribosomal binding sites.

How can I design efficient primers for amplifying and cloning the rps24 gene from T. rubripes samples?

When designing primers for T. rubripes rps24 amplification and cloning, consider the following methodological recommendations:

Primer Design Strategy:

• Target the complete coding sequence (CDS) of rps24, incorporating 15-20 nucleotides upstream of the start codon and downstream of the stop codon.

• Verify sequence accuracy using multiple Takifugu genome databases, as some genomic variations exist between database versions.

• Incorporate appropriate restriction sites, ensuring they are not present in the rps24 sequence.

• Add 3-6 nucleotides at the 5' end of restriction sites to enhance enzyme cutting efficiency.

• Consider adding sequences for tags and protease cleavage sites as needed.

Primer Optimization Parameters:

• Length: 18-30 nucleotides (excluding added features)

• GC content: 40-60%

• Tm: 55-65°C with minimal difference between forward and reverse primers

• Avoid sequences prone to secondary structure formation

• Verify specificity using BLAST against the T. rubripes genome

Example Primer Design for T. rubripes rps24:

Based on the close genetic relationship among Takifugu species, primers designed for T. rubripes may also amplify rps24 from related species like T. pseudommus and T. chinensis . When designing primers, the high genetic similarity (>99%) among these species should be considered . For species-specific amplification, target regions with known variations between species.

For cloning into expression vectors, incorporate vector-specific features:

• For pET-28a: 5'-CATATG (NdeI) at the start codon and 5'-CTCGAG (XhoI) before the stop codon

• For pGEX: 5'-GGATCC (BamHI) at the start codon and 5'-CTCGAG (XhoI) at the stop codon

PCR conditions should be optimized with a touchdown protocol, starting 5°C above the calculated Tm and gradually decreasing to the optimal annealing temperature.

What genetic variations exist in the rps24 gene across different Takifugu species and populations?

Research on genetic variations among Takifugu species provides insights into potential variations in the rps24 gene. While specific data on rps24 genetic variations were not directly presented in the search results, the broader patterns of genetic variation among Takifugu species offer valuable context.

Genetic analysis of closely related Takifugu species (T. rubripes, T. pseudommus, and T. chinensis) reveals remarkably low genetic differentiation . Population genetic studies using mitochondrial DNA (mtDNA) markers and simple sequence repeat (SSR) loci demonstrated that these three species show minimal genetic divergence and likely represent a single genetic population .

The genetic similarity among these species is evidenced by:

• Shared mtDNA haplotypes, with most individuals from all three species belonging to haplotype 1

• Low genetic diversity values (h=0.3743, π=0.006849)

• Minimal genetic differentiation in SSR analysis

For rps24 specifically, this suggests that functional coding regions are likely highly conserved across these species. Any variations that do exist might be concentrated in:

• Intronic regions

• 5' and 3' untranslated regions

• Promoter elements that regulate expression

Based on the broader genomic comparison between T. rubripes and T. pseudommus, which identified 1,438,490 SNPs, 377,741 deletions, and 356,158 insertions distributed across the genome , the rps24 gene may contain similar proportions of variations, with the majority likely occurring in non-coding regions.

How can I analyze the evolutionary conservation of rps24 across different fish species?

Analyzing evolutionary conservation of rps24 requires a systematic approach combining sequence analysis, structural modeling, and functional domain assessment:

Methodological Approach for Evolutionary Analysis:

• Sequence Collection and Alignment:

  • Retrieve rps24 sequences from diverse fish species spanning different taxonomic groups

  • Include representative species from various evolutionary distances

  • Align sequences using MUSCLE or MAFFT with optimization for small proteins

  • Manually curate alignments to ensure accuracy, particularly at indel boundaries

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood (RAxML or IQ-TREE)

  • Implement appropriate evolutionary models (JTT or LG for protein sequences)

  • Assess node support using bootstrap analysis (1000 replicates)

  • Root trees with appropriate outgroups (non-vertebrate rps24 sequences)

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify signatures of selection

  • Perform site-specific selection analysis using PAML or HyPhy

  • Identify sites under positive or purifying selection

• Functional Domain Conservation:

  • Map conserved domains using Pfam and InterPro

  • Quantify conservation scores for each amino acid position

  • Correlate conservation with known functional sites and interactions

For broader evolutionary comparisons, focus on regions known to interact with rRNA or other ribosomal proteins, as these typically show the highest conservation due to functional constraints.

What bioinformatic approaches are most effective for studying rps24 functional domains and interactions?

Several bioinformatic approaches can be employed to characterize rps24 functional domains and predict its interactions within the ribosomal complex:

1. Structural Prediction and Analysis:

• Homology modeling using SWISS-MODEL or I-TASSER based on crystal structures of ribosomal complexes

• Molecular dynamics simulations to predict flexible regions and binding interfaces

• Assessment of electrostatic surface potential to identify RNA-binding regions

2. Interaction Network Analysis:

• Prediction of protein-protein interactions using STRING database

• Identification of conserved interaction interfaces with other ribosomal proteins

• RNA-protein interaction prediction using catRAPID or RNABindR

3. Functional Domain Prediction:

• Identification of RNA-binding motifs using RBPmap or RNAProSite

• Prediction of post-translational modification sites using NetPhos, UbPred, or GPS

• Conservation analysis using ConSurf to map evolutionary conservation onto structural models

4. Regulatory Element Analysis:

• Prediction of transcription factor binding sites in the promoter region

• Identification of potential microRNA binding sites in 3' UTRs

• Analysis of alternative splicing patterns across different tissues

When analyzing ribosomal protein S24 in Takifugu species, it's important to consider the broader context of 40S ribosomal quality control. Research has shown that the abundance of 40S ribosomal proteins can be regulated through ubiquitylation, with the E3 ligase RNF10 and deubiquitinase USP10 playing antagonistic roles . Bioinformatic analysis of potential ubiquitylation sites in rps24 could provide insights into whether this protein might be similarly regulated.

The high genetic similarity between T. rubripes, T. pseudommus, and T. chinensis suggests that bioinformatic predictions made for one species would likely apply to all three, simplifying comparative analyses.

How can recombinant T. rubripes rps24 be used to study ribosomal quality control mechanisms?

Recombinant T. rubripes rps24 offers valuable research applications for investigating ribosomal quality control mechanisms, particularly within the context of the initiation RQC (iRQC) pathway that acts on 40S ribosomes during translation initiation .

Key Research Applications:

• Ubiquitylation Studies:

  • Recombinant rps24 can serve as a substrate in in vitro ubiquitylation assays with RNF10 E3 ligase

  • Site-directed mutagenesis of predicted ubiquitylation sites allows mapping of modification sites

  • Pull-down experiments can identify interaction partners in the ubiquitylation pathway

• 40S Assembly Investigation:

  • Fluorescently labeled recombinant rps24 can track 40S assembly dynamics

  • Competition assays with mutant versions can identify critical residues for assembly

  • Structure-function studies can define the role of rps24 in maintaining 40S integrity

• Quality Control Pathway Analysis:

  • Incorporation of recombinant rps24 into cellular systems allows tracking of its fate during stress

  • Comparison of wild-type and mutant rps24 processing can reveal quality control checkpoints

  • Interaction studies with components of the iRQC pathway can map the quality control network

Research has demonstrated that 40S ribosomal proteins like uS3 and uS5 are targeted for ubiquitylation by the E3 ligase RNF10, leading to selective reduction in 40S abundance . Using recombinant rps24 in similar experimental systems can determine whether this protein is also regulated through this mechanism.

The study of rps24 in ribosomal quality control is particularly relevant given the finding that 40S degradation occurs through an autophagy-independent mechanism , suggesting alternative degradation pathways that could be explored using recombinant protein tools.

What is the role of rps24 in the regulation of 40S ribosomal subunit abundance?

The regulation of 40S ribosomal subunit abundance is a complex process involving synthesis, assembly, and targeted degradation. While rps24-specific data from T. rubripes was not directly provided in the search results, insights can be drawn from studies on related 40S ribosomal proteins.

• Ubiquitylation-dependent control:

  • Specific 40S ribosomal proteins (uS3 and uS5) are targets for ubiquitylation by the E3 ligase RNF10

  • This ubiquitylation acts as a signal for degradation, reducing 40S abundance

  • The deubiquitinase USP10 counteracts this process, maintaining 40S levels

• Selective regulation of 40S vs. 60S:

  • RNF10 overexpression or USP10 knockout selectively reduces 40S abundance while 60S levels remain relatively unchanged

  • This suggests a specific quality control mechanism for 40S subunits that may involve rps24

• Post-translational rather than synthetic regulation:

  • Studies using metabolic pulse labeling demonstrated that 40S protein synthesis rates were actually increased in cells with enhanced 40S ubiquitylation

  • This indicates that reduced 40S abundance results from post-translational degradation rather than decreased synthesis

Based on these findings, rps24 likely participates in the regulated turnover of 40S subunits, potentially serving as either:

• A direct target for ubiquitylation in the quality control pathway

• A structural component whose modification affects the accessibility of other ribosomal proteins to ubiquitylation

• A sensor that helps distinguish properly assembled from defective 40S subunits

Understanding rps24's specific role would require direct experimental evidence using methods like site-directed mutagenesis, ubiquitylation assays, and pulse-chase experiments.

How might rps24 contribute to species-specific translation regulation in Takifugu?

While direct evidence for rps24's role in species-specific translation regulation in Takifugu is limited, several research-based hypotheses can be formulated based on the available data on ribosomal proteins and Takifugu genetics.

Potential Contributions of rps24 to Species-Specific Regulation:

• Subtle Sequence Variations and Functional Consequences:
  Despite the high genetic similarity among Takifugu species , even minor amino acid differences in rps24 could affect:

  • Interaction with species-specific mRNA features

  • Association with regulatory factors

  • Response to environmental stressors

• Differential Post-Translational Modifications:

  • Species-specific patterns of ubiquitylation or phosphorylation could tune translation efficiency

  • Different regulatory enzymes (like species-specific variants of RNF10 or USP10 ) might modify rps24 differently

  • These modifications could create species-specific translation profiles

• Integration in Takifugu-Specific Regulatory Networks:

  • Rps24 may interact with species-specific regulatory RNAs or proteins

  • Different expression patterns of rps24 across tissues could contribute to tissue-specific translation regulation

  • Environmental adaptations might manifest through altered rps24 function or regulation

The genetic evidence suggesting that T. rubripes, T. pseudommus, and T. chinensis represent a single genetic population indicates that any species-specific regulation would likely be subtle and perhaps involve:

• Epigenetic modifications rather than genetic differences

• Differential expression of rps24 variants through alternative splicing

• Interaction with species-specific regulatory factors

To investigate these possibilities, comparative studies of rps24 expression, modification patterns, and interaction partners across different Takifugu species would be required, along with functional assays examining translation efficiency and selectivity in different cellular contexts.

What are common challenges in expressing and purifying functional recombinant rps24?

Researchers frequently encounter several challenges when working with recombinant rps24 from T. rubripes:

Common Expression Challenges:

• Inclusion Body Formation:

  • Ribosomal proteins often form inclusion bodies due to their high basic amino acid content

  • Solution: Lower expression temperature (16°C), use solubility-enhancing tags (SUMO, MBP), or co-express with ribosomal RNA fragments

• Proteolytic Degradation:

  • Small ribosomal proteins can be rapidly degraded

  • Solution: Include multiple protease inhibitors, reduce expression time, optimize lysis conditions

• Low Expression Levels:

  • Codon bias between T. rubripes and expression hosts can limit yield

  • Solution: Use codon-optimized sequences, expression strains supplemented with rare tRNAs (like Rosetta™), or stronger promoters

Purification Challenges and Solutions:

• Co-purification of nucleic acids:

  • Ribosomal proteins naturally bind RNA, leading to contamination

  • Solution: Include high-salt washes (500 mM NaCl), treat with RNase A during purification, add polyethyleneimine precipitation step

• Aggregation during concentration:

  • Purified rps24 may aggregate at high concentrations

  • Solution: Include stabilizing agents (5-10% glycerol, 100-250 mM NaCl), avoid concentrating above 1-2 mg/mL

• Loss of functional conformation:

  • Recombinant expression may yield misfolded protein

  • Solution: Verify folding by circular dichroism, optimize refolding conditions, validate function through RNA binding assays

Table 2: Troubleshooting Guide for Recombinant rps24 Expression and Purification

When interpreting purification results, it's important to consider the findings from 40S ribosomal protein research, which show that these proteins can be subject to various modifications that affect their stability and interactions . Carefully monitoring these aspects during recombinant expression can help ensure functional protein production.

How can I validate the functional activity of purified recombinant rps24?

Validating the functional activity of recombinant T. rubripes rps24 requires multiple complementary approaches to assess its structural integrity and biological activity:

Structural Validation Methods:

• Circular Dichroism (CD) Spectroscopy:

  • Analyze secondary structure content and compare to predicted values

  • Monitor thermal stability (melting temperature)

  • Compare with CD spectra of other ribosomal proteins if available

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Verify monodispersity and expected molecular weight

  • Detect aggregation or oligomerization states

  • Assess sample homogeneity

• Limited Proteolysis:

  • Compare digestion patterns of recombinant versus native protein

  • Properly folded protein shows resistance to proteolysis at certain sites

  • Mass spectrometry analysis of fragments confirms structural domains

Functional Validation Methods:

• RNA Binding Assays:

  • Electrophoretic Mobility Shift Assay (EMSA) with rRNA fragments

  • Fluorescence anisotropy with labeled RNA

  • Surface Plasmon Resonance (SPR) to determine binding kinetics

• 40S Assembly Incorporation:

  • In vitro reconstitution assays with other 40S components

  • Fluorescently labeled rps24 to track incorporation

  • Pull-down assays to verify interactions with other ribosomal proteins

• Ubiquitylation Susceptibility:

  • In vitro ubiquitylation assays with RNF10 E3 ligase

  • Mass spectrometry to identify modification sites

  • Compare ubiquitylation patterns with those observed in cellular contexts

Research on 40S ribosomal proteins has shown that specific proteins (uS3 and uS5) undergo ubiquitylation that affects 40S abundance . Testing whether recombinant rps24 can be similarly modified provides valuable validation of its native-like conformation and functional role in quality control pathways.

The high genetic similarity among Takifugu species suggests that functional validation methods developed for one species should be applicable across related species, facilitating comparative studies.

How should I interpret conflicting results when studying rps24 in different experimental systems?

When encountering conflicting results in rps24 research across different experimental systems, a systematic approach to data interpretation is essential:

Methodological Framework for Resolving Conflicting Data:

• Experimental System Analysis:

  • Compare in vitro recombinant systems vs. cellular contexts

  • Evaluate differences between heterologous expression systems

  • Consider species-specific factors between T. rubripes and other model organisms

• Context-Dependent Function Assessment:

  • Analyze role of rps24 in different cellular compartments

  • Evaluate influence of stress conditions on results

  • Consider developmental or tissue-specific contexts

• Technical Variables Evaluation:

  • Assess differences in protein tags, fusion partners, or purification methods

  • Compare buffer conditions, particularly ionic strength and pH

  • Evaluate assay sensitivities and dynamic ranges

Specific Interpretative Approaches for Conflicting Data:

When studying rps24 ubiquitylation and its effect on 40S abundance, conflicting results may arise due to:

• Variable ubiquitylation machinery:

  • Different E3 ligases may target rps24 in different contexts

  • Studies of RNF10-mediated ubiquitylation suggest specificity for certain 40S proteins (uS3, uS5)

  • Opposing activities of ligases and deubiquitinases (like USP10) may vary across systems

• Differential 40S degradation pathways:

  • Research indicates that 40S degradation is autophagy-independent

  • Alternative degradation pathways may predominate in different cell types

  • The balance between synthesis and degradation may vary across systems

• Species-specific variations:

  • Despite high genetic similarity among Takifugu species , subtle differences may exist

  • Comparison with data from distantly related species requires careful interpretation

  • Evolutionary conservation analysis can help distinguish fundamental vs. species-specific functions

When encountering contradictory results, consider designing experiments that:

• Directly compare different systems under identical conditions

• Systematically isolate variables to identify critical factors

• Utilize genetic approaches (knockouts, mutations) to validate biochemical findings

• Incorporate multiple complementary techniques to build a coherent model

The post-translational regulation of 40S ribosomal proteins appears to involve complex interplay between synthesis, ubiquitylation, and degradation , suggesting that seemingly conflicting results may actually reflect different aspects of this multi-layered regulatory system.