Recombinant Strongylocentrotus purpuratus 40S ribosomal protein S15a (RPS15A)

What is RPS15A and what is its fundamental function in Strongylocentrotus purpuratus?

RPS15A (Ribosomal Protein S15A) is a highly conserved 40S ribosomal protein that plays an essential role in cellular protein synthesis. In eukaryotes, including S. purpuratus, it functions primarily by promoting mRNA/ribosome interactions during translation through interactions with the cap-binding subunit of eukaryotic initiation factor 4F (eIF-4F) . This protein is critical for the process of eukaryotic protein biosynthesis and cellular survival . In sea urchin models like S. purpuratus, RPS15A likely maintains these conserved translational functions while potentially exhibiting species-specific regulatory mechanisms during development.

The functional significance of RPS15A extends beyond standard translation. Research has shown that in yeast, G1/S cell cycle phase arrest induced by cdc33 (encoding eIF-4F in yeast) mutation could be reversed by RPS15A over-expression, suggesting it plays a critical role in cell cycle transition . This connection to cell cycle regulation has significant implications for understanding both normal development and pathological states.

How conserved is RPS15A across species from sea urchins to humans?

RPS15A demonstrates remarkable evolutionary conservation across diverse species, reflecting its fundamental importance in cellular function. Human RPS15A maps to chromosome 16p12.3 locus and has been characterized as "a highly conserved cellular gene" . While the search results don't provide specific sequence identity percentages between S. purpuratus and human RPS15A, the functional domains responsible for mRNA/ribosome interactions are likely to show the highest conservation.

The conservation of RPS15A across species suggests that findings from human studies may have relevance to understanding S. purpuratus RPS15A function, particularly regarding:

• Core translational mechanisms

• Interactions with initiation factors

• Structural components essential for ribosome assembly

• Potential extra-ribosomal functions

This evolutionary conservation makes S. purpuratus RPS15A a valuable model for studying fundamental aspects of translation that may apply across eukaryotic species.

What techniques are most effective for recombinant expression and purification of S. purpuratus RPS15A?

While the search results don't specifically address purification of S. purpuratus RPS15A, successful methodologies for human RPS15A can be adapted. Based on established protocols for ribosomal proteins, the following approach is recommended:

Expression System Selection:

• Bacterial expression (E. coli BL21): Suitable for structural studies, provides high yield but may lack post-translational modifications

• Yeast expression systems: Better for functional studies requiring eukaryotic folding mechanisms

• Insect cell expression: Recommended for complex eukaryotic proteins requiring specific modifications

Purification Protocol:

• Clone the S. purpuratus RPS15A gene from genomic DNA or cDNA library

• Insert into an expression vector with appropriate affinity tags (6xHis or GST)

• Transform into chosen expression system

• Induce expression (IPTG for bacterial systems)

• Lyse cells under native or denaturing conditions based on solubility

• Purify using affinity chromatography followed by size exclusion chromatography

• Verify purity using SDS-PAGE and Western blotting

• Confirm identity with mass spectrometry

For functional studies, it's critical to verify that the recombinant protein maintains its ribosome-binding capacity through in vitro translation assays.

What is the gene structure and protein domain organization of S. purpuratus RPS15A?

Based on the conserved nature of ribosomal proteins and information about human RPS15A, the domain structure of S. purpuratus RPS15A likely includes:

Gene Structure:

• Compact genomic organization typical of ribosomal proteins

• Potential presence of introns that may be involved in regulation of expression

• Promoter elements responsive to growth factors and developmental signals

Protein Domains:

• N-terminal region: Often contains signal sequences for ribosomal localization

• Central RNA-binding domain: Critical for interaction with ribosomal RNA

• Protein-protein interaction domains: For assembly with other ribosomal components

• Regions that interact with translation initiation factors like eIF-4F

• Potential regulatory domains subject to post-translational modifications

The functional significance of these domains can be investigated through targeted mutagenesis and domain swap experiments between species variants of RPS15A.

How can RPS15A knockdown be achieved in S. purpuratus and what phenotypes might result?

Based on successful approaches with human cell lines, several methodologies can be adapted for S. purpuratus RPS15A knockdown:

Knockdown Methodologies:

Research on human cancer cells provides insights into potential phenotypes following RPS15A knockdown. Studies demonstrated that knockdown of RPS15A significantly suppressed cell proliferation and colony formation in cancer cell lines, and induced cell cycle arrest at G0/G1 phase . Gene expression analysis revealed that RPS15A knockdown activated the p53 signaling pathway, upregulating p53 and p21 expression while downregulating CDK1 expression .

In S. purpuratus embryos, similar knockdown might result in:

• Developmental delays or arrests, particularly in rapidly dividing cells

• Altered cell cycle progression during embryogenesis

• Potential activation of apoptotic pathways

• Dysregulation of protein synthesis affecting morphogenesis

What signaling pathways interact with RPS15A in model organisms?

Research in human cancer models has identified several important pathway interactions that may be conserved in S. purpuratus:

P53 Signaling Pathway:
Studies in human cancer cells have demonstrated that RPS15A knockdown activates the p53 signaling pathway . This activation results in increased expression of p53 and its downstream target p21, while decreasing expression of CDK1, which collectively leads to cell cycle arrest and potential apoptosis . Since S. purpuratus possesses p53 homologs, similar regulatory mechanisms likely exist.

Cell Cycle Regulation:
RPS15A has been implicated in cell cycle transition, particularly at the G1/S checkpoint. In yeast, RPS15A overexpression rescued G1/S arrest induced by eIF-4F mutation , suggesting a conserved role in cell cycle progression that may extend to sea urchins.

Additional Potential Pathway Interactions:

• mTOR signaling pathway (regulating protein synthesis)

• Stress response pathways (responding to translational defects)

• Developmental signaling cascades specific to embryogenesis

These pathway interactions could be investigated in S. purpuratus using phosphoproteomic analysis following RPS15A manipulation.

How does RPS15A expression change during embryonic development in S. purpuratus?

While the search results don't provide specific data on developmental expression patterns of RPS15A in S. purpuratus, based on knowledge of ribosomal proteins and sea urchin development, we can predict:

Expected Developmental Expression Pattern:

Experimental validation of these patterns would require:

• Quantitative PCR across developmental time points

• In situ hybridization to visualize spatial expression patterns

• Immunohistochemistry with RPS15A-specific antibodies

• Translational reporter constructs to monitor activity

Understanding the developmental regulation of RPS15A could provide insights into its role in normal development and potential implications for developmental disorders.

What is the role of post-translational modifications in regulating S. purpuratus RPS15A function?

Post-translational modifications (PTMs) likely play crucial roles in regulating RPS15A function, although specific modifications in S. purpuratus RPS15A are not detailed in the search results. Based on studies of ribosomal proteins in other organisms, several PTMs may be significant:

Potential Regulatory Modifications:

These modifications might be developmentally regulated in S. purpuratus or respond to cellular stresses, potentially providing a mechanism for rapid adaptation of translational machinery to changing environmental or developmental conditions.

How do cancer-associated alterations in human RPS15A compare to evolutionary variations in S. purpuratus RPS15A?

Research has identified RPS15A as highly expressed in human colorectal cancer and lung adenocarcinoma, with expression levels correlating with poor prognosis . This presents an interesting opportunity for comparative analysis:

Comparative Analysis Framework:

• Identification of conserved domains between human and S. purpuratus RPS15A

• Mapping of cancer-associated mutations/alterations onto protein structure

• Analysis of whether evolutionary divergent regions correspond to cancer-susceptible domains

• Determination if cancer-associated changes affect conserved or variable regions

Such analysis could provide insights into:

• Whether regions of RPS15A that show natural variation between humans and sea urchins differ from regions altered in cancers

• If evolutionarily conserved regions represent functionally critical domains that cannot tolerate mutation

• Potential identification of RPS15A domains that might be safely targeted for cancer therapies

High RPS15A expression in human colorectal cancer correlates with several clinical features including older age (P=0.035), not receiving preoperative neoadjuvant treatment (P=0.048), higher primary pN stage (P=0.007) and slightly more synchronous distant metastases (P=0.058) . Understanding the structural basis for these associations could inform both evolutionary biology and cancer research.

What are the optimal laboratory conditions for studying recombinant S. purpuratus RPS15A?

For effective study of recombinant S. purpuratus RPS15A, consider the following optimized conditions:

Expression Systems Comparison:

Buffer Optimization:

• Extraction: PBS pH 7.4 with protease inhibitors

• Purification: Gradient of imidazole (20-250 mM) for His-tagged proteins

• Storage: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

Quality Control Metrics:

• Purity: >95% by SDS-PAGE

• Identity: Confirmation by mass spectrometry

• Activity: Ribosome binding assay

• Stability: Thermal shift assay to determine optimal buffer conditions

These parameters should be systematically optimized for each specific research application.

What antibodies and detection methods are effective for studying S. purpuratus RPS15A?

While the search results don't mention specific antibodies for S. purpuratus RPS15A, several approaches can be considered:

Antibody Development and Selection:

• Cross-reactivity testing of commercial anti-human RPS15A antibodies

• Custom antibody production using:

  • Synthetic peptides from conserved regions

  • Full-length recombinant S. purpuratus RPS15A

• Epitope tagging (HA, FLAG, GFP) of recombinant RPS15A for detection with tag-specific antibodies

Detection Methods Optimization:

Validation would require Western blotting against sea urchin tissue extracts and appropriate negative controls to confirm specificity.

How can CRISPR/Cas9 gene editing be optimized for modifying RPS15A in S. purpuratus?

CRISPR/Cas9 gene editing in sea urchins requires specific optimization for targeting RPS15A:

CRISPR/Cas9 Protocol for S. purpuratus RPS15A:

• Guide RNA Design:

  • Target conserved exons using algorithms that minimize off-target effects

  • Design multiple gRNAs targeting different regions of the gene

  • Test gRNA efficiency using in vitro cleavage assays

• Delivery Method:

  • Microinjection of CRISPR/Cas9 components into fertilized eggs

  • Optimization of Cas9 concentration (50-300 ng/μl) and gRNA (25-100 ng/μl)

  • Consider Cas9 protein with pre-complexed gRNA rather than mRNA

• Editing Strategies:

  • Knockout: Design gRNAs to create frameshift mutations

  • Knock-in: Include homology-directed repair template with desired mutation

  • Conditional systems: Consider inducible Cas9 expression for developmental studies

• Screening Methods:

  • T7 Endonuclease I assay for initial editing efficiency assessment

  • RFLP analysis if edit creates/destroys restriction sites

  • Direct sequencing of PCR products from individual embryos

  • Protein expression analysis by Western blot

Special considerations for ribosomal proteins like RPS15A include the potential lethality of complete knockouts, necessitating conditional approaches or heterozygous edits.

What are the best experimental controls when studying RPS15A function in sea urchin models?

Robust controls are essential for reliable interpretation of RPS15A functional studies:

Essential Control Types:

Research on human cancer cells has effectively used control shRNA lentiviruses (Lv-shCon) alongside RPS15A-specific shRNA (Lv-shRPS15A) to demonstrate specific effects of RPS15A knockdown . Similar approaches would be valuable in sea urchin studies.

How does RPS15A contribute to the p53 signaling pathway?

Research in human cancer cells has revealed important connections between RPS15A and p53 signaling:

Key Molecular Interactions:

Gene expression profile microarray analysis revealed that the p53 signaling pathway was activated in RPS15A-knockdown cancer cells . Further studies confirmed that RPS15A knockdown specifically:

• Upregulated p53 expression

• Increased p21 expression (a downstream target of p53)

• Decreased CDK1 expression

These changes collectively contribute to cell cycle arrest at G0/G1 phase and potential induction of apoptosis . The mechanism may involve ribosomal stress signaling, where alterations in ribosome assembly trigger p53 activation via MDM2 inhibition.

In S. purpuratus, which possesses p53 homologs, similar pathway interactions likely exist, though developmental contexts may introduce additional regulatory mechanisms. This relationship between RPS15A and p53 signaling has significant implications for both developmental biology and cancer research.

What techniques are most effective for analyzing RPS15A-dependent translational regulation?

To investigate the impact of RPS15A on translation in S. purpuratus or other models:

Advanced Methodological Approaches:

• Polysome Profiling:

  • Separates mRNAs based on ribosome association

  • Can identify changes in global translation efficiency following RPS15A manipulation

  • Requires careful optimization of sucrose gradient conditions

• Ribosome Footprinting:

  • Provides nucleotide-resolution map of ribosome positions on mRNAs

  • Can identify specific transcripts affected by RPS15A alterations

  • Requires deep sequencing and specialized bioinformatic analysis

• SILAC or TMT Mass Spectrometry:

  • Quantifies changes in newly synthesized proteins

  • Distinguishes translational from transcriptional effects

  • Requires metabolic labeling or chemical tagging

• Reporter Constructs:

  • Luciferase or fluorescent protein reporters with various 5'UTRs

  • Tests effect of RPS15A on specific mRNA translation

  • Can be adapted for in vivo studies in sea urchin embryos

Research has shown that RPS15A promotes mRNA/ribosome binding in translation through interactions with the cap-binding subunit of eukaryotic initiation factor 4F (eIF-4F) , making it particularly important to examine cap-dependent translation initiation.

How can S. purpuratus RPS15A be used as a model to study ribosomal protein involvement in human diseases?

S. purpuratus RPS15A offers several advantages as a model for studying ribosomal protein functions relevant to human diseases:

Research Applications:

• Developmental Context:

  • Sea urchin embryos provide an excellent model for studying development

  • Can reveal how RPS15A dysfunction affects embryogenesis

  • May provide insights into developmental disorders linked to ribosomal proteins

• Cancer Biology:

  • RPS15A is implicated in colorectal cancer and lung adenocarcinoma

  • Sea urchin models allow study of RPS15A in normal developmental context

  • Comparison may reveal how normal functions are subverted in cancer

• Experimental Advantages:

  • Sea urchin embryos are transparent and develop externally, facilitating imaging

  • Genetic manipulations can be performed relatively easily

  • Effects on cell proliferation and apoptosis can be studied in a non-cancer context

What are the challenges and solutions in developing therapeutic approaches targeting RPS15A?

Given the association of RPS15A with cancer progression, it represents a potential therapeutic target, though significant challenges exist:

Therapeutic Targeting Considerations:

Using S. purpuratus as a model system could help identify the threshold levels of RPS15A inhibition that affect abnormal growth without disrupting normal cellular functions.