Recombinant Coturnix coturnix japonica 40S ribosomal protein S17 (RPS17)

What is the structural and functional role of 40S ribosomal protein S17 in protein synthesis?

40S ribosomal protein S17 is a component of the small 40S ribosomal subunit that plays a critical role in the translation machinery. Ribosomes, which catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit, together comprising 4 RNA species and approximately 80 structurally distinct proteins . RPS17 belongs to the S17E family of ribosomal proteins and is primarily located in the cytoplasm . As part of the 40S subunit, it contributes to the structural integrity of the ribosome and participates in the translation initiation process, where it may interact with mRNA during scanning and start codon recognition.

How does avian RPS17 compare to mammalian homologs?

While the search results don't provide specific comparisons between avian and mammalian RPS17, ribosomal proteins are generally highly conserved across species due to their fundamental role in protein synthesis. The functional domains of RPS17 likely maintain high sequence similarity across vertebrates, though species-specific variations may exist. Japanese quail (Coturnix japonica) RPS17 would share structural features with its homologs in other species, including the human version which belongs to the S17E family of ribosomal proteins . Researchers should perform sequence alignment analyses to identify conserved domains when designing experiments that rely on structural or functional homology.

What methods are recommended for initial characterization of recombinant RPS17?

For initial characterization of recombinant Coturnix coturnix japonica RPS17, researchers should employ a systematic approach:

• Protein expression verification: Use SDS-PAGE and Western blotting with antibodies specific to RPS17 or attached tags (His, FLAG, etc.)

• Mass spectrometry analysis: Confirm protein identity and detect potential post-translational modifications

• Circular dichroism spectroscopy: Assess secondary structure content and proper folding

• Functional assays: Test binding to rRNA and other ribosomal proteins to verify biological activity

• Thermal shift assays: Evaluate protein stability under different buffer conditions

These methods provide a foundation for further experimental work, ensuring the recombinant protein exhibits characteristics consistent with native RPS17.

How can recombinant RPS17 be used to study ribosomal quality control pathways?

Recombinant RPS17 can serve as a valuable tool for investigating ribosomal quality control (RQC) pathways, particularly in the context of the newly identified initiation RQC (iRQC) that acts on 40S ribosomes during translation initiation . Researchers could:

• Develop in vitro ubiquitylation assays: Using purified 40S subunits containing recombinant RPS17 to study site-specific ubiquitylation patterns

• Create modified RPS17 variants: Engineer recombinant RPS17 with mutations at potential ubiquitylation sites to study their impact on 40S stability and degradation

• Study protein-protein interactions: Use recombinant RPS17 as bait to identify novel interacting partners involved in ribosome quality control, such as E3 ligases similar to RNF10 which regulates other 40S proteins (uS3 and uS5)

• Design reconstitution experiments: Incorporate recombinant RPS17 into 40S subunits to study how its modifications influence ribosome assembly and quality control

Recent research has identified distinct ribosomal quality control pathways, including iRQC that specifically monitors 40S ribosomes during translation initiation . Studying how RPS17 participates in these pathways could reveal critical insights into translation regulation.

What experimental approaches are recommended for studying post-translational modifications of RPS17?

Post-translational modifications (PTMs) of ribosomal proteins enable rapid and dynamic regulation of protein biogenesis . To study PTMs of recombinant Coturnix coturnix japonica RPS17:

• Mass spectrometry-based proteomics:

  • Use bottom-up proteomics to identify modification sites

  • Apply SILAC labeling to quantify modification stoichiometry under different conditions

  • Perform top-down proteomics to analyze the combination of multiple modifications

• Site-specific mutagenesis:

  • Generate recombinant RPS17 variants with mutations at potential modification sites

  • Assess the impact of these mutations on ribosome assembly and function

• PTM-specific antibodies:

  • Develop antibodies recognizing specific PTMs on RPS17

  • Use these for Western blotting and immunoprecipitation experiments

• In vitro modification assays:

  • Reconstitute the modification reaction using purified enzymes and recombinant RPS17

  • Study the kinetics and specificity of the modification process

The study of ribosomal protein ubiquitylation has revealed its critical role in quality control pathways . Similar approaches could be applied to identify and characterize PTMs on RPS17 and their functional significance.

How can differential expression of RPS17 across developmental stages be effectively analyzed?

Based on findings related to developmental stage-related differences in Japanese quail , researchers can employ several approaches to study RPS17 expression across development:

• RNA-Seq analysis:

  • Compare RPS17 transcript levels across different developmental stages

  • Analyze in conjunction with other ribosomal proteins to identify coordinated expression patterns

• Proteomics approaches:

  • Use SILAC or TMT labeling to quantify RPS17 protein levels across developmental stages

  • Compare post-translational modification patterns at different stages

• Tissue-specific expression analysis:

  • Examine RPS17 expression in different tissues throughout development

  • Use laser capture microdissection to isolate specific cell types for analysis

• Reporter systems:

  • Create transgenic models with RPS17 promoter-driven reporters to visualize expression patterns

  • Use CRISPR/Cas9 to tag endogenous RPS17 for live imaging

Research in Japanese quail has shown that the number of differentially expressed genes is higher in embryos than in adults, suggesting dynamic regulation during development . This approach could be extended to specifically study RPS17 expression patterns.

What are the optimal expression systems for producing recombinant Coturnix coturnix japonica RPS17?

When selecting an expression system for recombinant RPS17 production, researchers should consider several factors:

For basic structural studies, E. coli systems may be sufficient, while studies focusing on PTMs would benefit from mammalian expression systems. The choice should be guided by the specific research questions being addressed.

What purification strategies are most effective for recombinant RPS17?

A multi-step purification strategy is recommended for obtaining high-purity recombinant RPS17:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged RPS17

  • Glutathione affinity chromatography for GST-tagged RPS17

• Intermediate purification:

  • Ion exchange chromatography to separate based on charge differences

  • Heparin affinity chromatography, exploiting RPS17's RNA-binding properties

• Polishing:

  • Size exclusion chromatography to remove aggregates and achieve high purity

  • Reverse phase HPLC for final purification if needed

• Quality control:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Dynamic light scattering to assess homogeneity

  • Mass spectrometry to confirm molecular weight and modifications

The purification protocol should be optimized based on the expression system used and the intended application of the purified protein.

How can recombinant RPS17 be used to study sex-specific differences in ribosome function?

Based on findings showing sex-related differences in Japanese quail gene expression , researchers can design experiments to investigate sex-specific aspects of ribosome function using recombinant RPS17:

• Comparative analysis of RPS17 modifications:

  • Isolate native RPS17 from male and female quail tissues

  • Compare PTM patterns with mass spectrometry

  • Recreate sex-specific modifications on recombinant RPS17 for functional studies

• Sex-specific interactome analysis:

  • Use recombinant RPS17 as bait to capture interacting partners from male and female tissue lysates

  • Identify differential binding partners through mass spectrometry

  • Validate interactions using techniques like co-immunoprecipitation and proximity ligation assays

• Hormonal regulation studies:

  • Investigate how sex hormones influence RPS17 expression and modification

  • Assess the impact of hormone exposure on ribosome assembly and function

• Tissue-specific translation regulation:

  • Compare RPS17 incorporation into ribosomes across tissues with sex-dimorphic functions

  • Analyze differential translation efficiency using ribosome profiling

Research has shown that male and female Japanese quail exhibit different patterns of gene expression in response to environmental factors , suggesting sex-specific regulation mechanisms that may extend to ribosomal proteins like RPS17.

How can structural studies of recombinant RPS17 contribute to understanding ribosome assembly?

Structural characterization of recombinant RPS17 can provide valuable insights into ribosome assembly mechanisms:

• X-ray crystallography and cryo-EM studies:

  • Determine the high-resolution structure of isolated RPS17

  • Compare with the structure of RPS17 within the assembled ribosome

  • Identify conformational changes that occur during assembly

• Interaction interface mapping:

  • Use hydrogen-deuterium exchange mass spectrometry to identify regions involved in protein-protein and protein-RNA interactions

  • Perform cross-linking studies to capture transient interactions during assembly

• In vitro assembly reconstitution:

  • Use purified recombinant RPS17 in ribosome assembly assays

  • Monitor assembly intermediates using analytical ultracentrifugation and electron microscopy

  • Identify the step at which RPS17 incorporates into the forming 40S subunit

• Impact of mutations:

  • Generate RPS17 variants with mutations at key interfaces

  • Assess their impact on assembly efficiency and kinetics

  • Correlate structural features with assembly checkpoints

These approaches can help elucidate how RPS17 contributes to 40S ribosomal subunit assembly and stability, complementing studies that have identified quality control mechanisms for ribosomal proteins .

What methods can be used to study the role of RPS17 in ribosomal quality control pathways?

Building on recent discoveries about ribosomal quality control pathways , researchers can investigate RPS17's potential role using the following approaches:

• Ubiquitylation site mapping:

  • Identify potential ubiquitylation sites on RPS17 using mass spectrometry

  • Generate recombinant RPS17 with mutations at these sites to study their functional significance

  • Perform in vitro ubiquitylation assays with purified E3 ligases like RNF10

• Degradation pathway analysis:

  • Study the fate of ubiquitylated RPS17 using pulse-chase experiments

  • Determine if RPS17 ubiquitylation leads to 40S degradation similar to what has been observed for other ribosomal proteins

  • Investigate if this occurs through autophagy-independent mechanisms as seen with other 40S proteins

• Stress response studies:

  • Examine how various cellular stresses affect RPS17 modification patterns

  • Determine if RPS17 participates in stress-specific ribosome remodeling

  • Compare with known stress responses involving other ribosomal proteins like uS3 and uS5

• Interaction with quality control factors:

  • Identify potential interactions between RPS17 and known ribosomal quality control factors

  • Investigate if RPS17 serves as a sensor for ribosome integrity

  • Study how these interactions change under different cellular conditions

Recent research has identified the iRQC pathway that specifically monitors 40S ribosomes during translation initiation . Understanding how RPS17 participates in this or similar pathways could provide important insights into translation regulation mechanisms.

How can researchers address solubility issues with recombinant RPS17?

Ribosomal proteins can present solubility challenges due to their highly basic nature and tendency to bind nucleic acids. The following strategies can help improve recombinant RPS17 solubility:

• Optimization of expression conditions:

  • Lower the expression temperature (16-18°C)

  • Use weaker promoters to slow expression rate

  • Optimize induction parameters (inducer concentration, timing, duration)

• Buffer optimization:

  • Include higher salt concentrations (300-500 mM) to disrupt ionic interactions

  • Add nuclease treatment steps to remove bound nucleic acids

  • Test different pH conditions (typically pH 7.5-8.5 works best for ribosomal proteins)

  • Include stabilizing additives such as glycerol (10-20%) or mild detergents

• Fusion partner strategies:

  • Use solubility-enhancing tags (SUMO, MBP, GST)

  • Ensure tag removal options that don't compromise protein stability

  • Position tags to minimize interference with folding

• Co-expression approaches:

  • Co-express with ribosomal RNA segments that naturally bind RPS17

  • Co-express with chaperone proteins to assist folding

  • Consider co-expression with natural binding partners

These strategies have proven effective for other ribosomal proteins and can be adapted for RPS17 based on specific experimental observations.

What are the critical quality control checkpoints for recombinant RPS17 research?

To ensure reliable results in experiments using recombinant RPS17, researchers should implement these quality control checkpoints:

Implementing these checkpoints helps ensure that experimental outcomes reflect true biological properties rather than artifacts from protein preparation.