RPS13 Human

What is the molecular structure and basic characterization of human RPS13?

Human RPS13 is a 17 kDa protein containing 151 amino acids that belongs to the S15P family of ribosomal proteins . It functions as a component of the 40S ribosomal subunit, participating in protein synthesis by facilitating the assembly of ribosomes.

The protein is located primarily in the cytoplasm and contains multiple phosphorylated residues that may regulate its function . When studying RPS13, researchers should note that commercial recombinant versions often include tags (such as His-tags) that extend the actual molecular weight to approximately 19.6 kDa .

Structurally, RPS13 (similar to its prokaryotic homolog S15) binds to the central domain of ribosomal RNA and promotes the binding of neighboring proteins during ribosomal subunit assembly . For ribosomal localization studies, fluorescent fusion proteins (such as RPS13-eCFP) typically demonstrate nuclear and nucleolar localization when overexpressed in eukaryotic cells .

How is the RPS13 gene organized and what regulatory elements are present?

The human RPS13 gene contains multiple introns, with intron 1 showing remarkable evolutionary conservation across mammals and birds, suggesting important regulatory functions . Comparative genomic analysis reveals this conservation extends to organisms including Canis familiaris, Rattus norvegicus, Mus musculus, and Gallus gallus .

Similar to other ribosomal protein genes, RPS13 has multiple processed pseudogenes dispersed throughout the genome, a common feature that complicates genomic analysis of ribosomal proteins . The gene is co-transcribed with two U14 small nucleolar RNA genes located in its third and fifth introns .

For experimental analysis of RPS13 gene regulation, researchers should consider:

• The presence of specific regulatory elements in intron 1

• The differential conservation pattern across introns

• The potential for autoregulatory feedback mechanisms

• The impact of pseudogenes on experimental design and analysis

What are the established functions of RPS13 in cellular biology?

Human RPS13 serves multiple roles in cellular biology:

Primary Functions:

• Component of the 40S ribosomal subunit essential for protein synthesis

• Involved in the early steps of ribosome assembly (similar to its bacterial ortholog)

• Participates in the initial stage of pre-18S rRNA processing (based on yeast homolog studies)

Extraribosomal Functions:

• Potential role in multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

• Interaction with PDCD4 (programmed cell death protein 4), suggesting involvement in regulatory pathways beyond translation

• Autoregulation of its own gene expression through a feedback mechanism at the pre-mRNA splicing level

For experimental validation of these functions, researchers commonly employ knockdown/knockout approaches, coupled with phenotypic analysis and ribosome profiling to distinguish between translational and extraribosomal effects.

How can I effectively study RPS13 autoregulation at the pre-mRNA splicing level?

To investigate the autoregulatory mechanism of RPS13, the following experimental approaches are recommended:

Minigene Constructs:

• Create reporter gene constructs containing the coding sequence of RPS13 with and without intron 1

• Transfect these constructs into appropriate cell lines (e.g., HEK 293 cells)

• Measure expression levels using fluorescent reporters like eCFP

• Compare expression levels between constructs with and without intron 1

In Vitro Splicing Assays:

• Generate 32P-labeled RNA transcripts containing exon-intron boundaries of RPS13

• Incubate with purified RPS13 protein under appropriate buffer conditions

• Analyze splicing efficiency through gel electrophoresis

• Include control RNA transcripts (e.g., fragments of rpS17 and rpS26 pre-mRNAs)

RNA-Protein Binding Studies:

• Perform immunoprecipitation using purified antibodies against RPS13

• Use protein G-sepharose resin for antibody immobilization

• Analyze RNA-protein complexes to identify binding regions

• Conduct ribonuclease protection assays to map the exact binding sites near splice junctions

The experimental evidence from these approaches should demonstrate that:

• Intron 1 presence reduces expression approximately four-fold in transfection studies

• RPS13 protein inhibits excision of intron 1 from its own pre-mRNA in vitro

• The protein binds specifically to sequences near the 5′ and 3′ splice sites of intron 1

• The binding confers protection against ribonuclease cleavage at these sites

What are the best approaches for purification and characterization of recombinant RPS13?

For successful purification and characterization of recombinant RPS13, consider the following methodological approaches:

Expression Systems:

• E. coli: Commonly used for high-yield production; protein is expressed as a single, non-glycosylated polypeptide chain

• HEK293T cells: Preferred for human post-translational modifications; available as lysates for certain applications

Purification Strategy:

• Express RPS13 with an affinity tag (e.g., His-tag at N-terminus)

• Use proprietary chromatographic techniques for initial purification

• Achieve >80% purity as determined by SDS-PAGE analysis

• Consider buffer optimization (e.g., 20mM Tris-HCl buffer pH 8.0, 0.2M NaCl, 50% glycerol, and 2mM DTT)

Characterization Methods:

• SDS-PAGE for purity assessment and molecular weight confirmation

• Western blotting using specific antibodies

• Mass spectrometry for exact mass determination and identification of post-translational modifications

• Functional assays to verify RNA binding activity

Storage Recommendations:

• Store at 4°C if using within 2-4 weeks

• For longer periods, store frozen at -20°C

• Consider adding carrier protein (0.1% HSA or BSA) for long-term storage

• Avoid repeated freeze-thaw cycles

What experimental designs are appropriate for studying RPS13's role in ribosome assembly?

To investigate RPS13's role in ribosome assembly, researchers should consider these experimental approaches:

Ribosome Profiling:

• Use sucrose gradient ultracentrifugation to separate ribosomal subunits, monosomes, and polysomes

• Analyze fractions for presence of RPS13 using western blotting

• Compare profiles between normal and RPS13-depleted cells

Cryo-EM Analysis:

• Purify ribosomal subunits from cells with normal or altered RPS13 expression

• Use cryo-electron microscopy to visualize structural changes

• Map the position of RPS13 within the 40S subunit

• Identify interacting partners within the ribosomal complex

In Vivo Assembly Assays:

• Create RPS13 variants with specific mutations based on structural data

• Express these variants in cells depleted of endogenous RPS13

• Analyze effects on ribosome assembly using sucrose gradients

• Monitor pre-rRNA processing by northern blotting

SILAC-Based Interaction Studies:

• Use stable isotope labeling with amino acids in cell culture (SILAC)

• Immunoprecipitate RPS13-containing complexes at different stages of ribosome assembly

• Identify stage-specific interaction partners using mass spectrometry

For proper experimental design, researchers should include appropriate controls and validate findings using multiple complementary approaches. When analyzing data, consider both statistical significance and biological relevance of observed changes.

How can I investigate the extraribosomal functions of RPS13 in cancer biology?

To explore RPS13's potential extraribosomal functions in cancer, particularly its reported role in multidrug resistance in gastric cancer , consider the following experimental approaches:

Gene Expression Manipulation:

• Use siRNA/shRNA for transient or stable knockdown of RPS13 in cancer cell lines

• Employ CRISPR-Cas9 for complete knockout studies

• Create overexpression systems with inducible promoters

• Develop rescue experiments with wild-type and mutant RPS13 variants

Functional Assays:

• Drug sensitivity testing using dose-response curves in cells with altered RPS13 expression

• Apoptosis assays (e.g., Annexin V/PI staining, caspase activity) to evaluate RPS13's reported anti-apoptotic effects

• Cell cycle analysis to identify potential cell cycle regulatory roles

• Migration and invasion assays to assess effects on metastatic potential

Molecular Mechanism Investigation:

• Immunoprecipitation followed by mass spectrometry to identify cancer-specific interaction partners

• ChIP-seq or CLIP-seq to identify DNA/RNA binding targets

• Phosphoproteomics to characterize RPS13 post-translational modifications in cancer contexts

• Pathway analysis to determine signaling networks affected by RPS13 modulation

In Vivo Models:

• Xenograft studies using cancer cells with manipulated RPS13 expression

• Patient-derived xenografts to validate findings in more clinically relevant models

• Analysis of cancer patient samples for RPS13 expression correlation with clinical outcomes

When designing these experiments, researchers should carefully separate ribosomal from extraribosomal functions by including appropriate controls (e.g., other ribosomal proteins) and considering the use of RPS13 mutants that specifically disrupt extraribosomal functions while maintaining ribosomal incorporation.

What approaches are effective for studying the evolutionary conservation of RPS13 regulation?

To investigate the evolutionary conservation of RPS13 regulation across species, particularly focusing on the highly conserved intron 1 region , implement these methodological approaches:

Comparative Genomics:

• Perform multi-species alignment of RPS13 gene sequences, particularly focusing on intron 1 regions

• Use tools like Evolutionary Conserved Regions (ECR) Browser to visualize conservation patterns

• Identify specific regulatory elements through motif discovery algorithms

• Analyze selective pressure on different regions using dN/dS ratio calculations

Functional Conservation Testing:

• Create hybrid minigene constructs containing intron 1 from different species

• Test the autoregulatory capacity of these constructs in human cell lines

• Perform cross-species binding assays with purified RPS13 proteins and heterologous pre-mRNAs

• Analyze conservation of autoregulation mechanisms in model organisms

Experimental Validation Table:

Evolutionary Analysis:

• Reconstruct the evolutionary history of RPS13 regulation

• Correlate regulatory changes with species-specific translational requirements

• Compare conservation patterns of RPS13 with other ribosomal proteins

• Analyze co-evolution with interacting partners (e.g., PDCD4)

For comprehensive analysis, integrate findings from comparative genomics with experimental validation and consider the broader context of ribosome evolution and adaptation.

How does RPS13 interact with PDCD4 and what are the functional implications?

The interaction between RPS13 and programmed cell death protein 4 (PDCD4) has been documented , but the functional implications remain an area for active research. To investigate this interaction:

Interaction Characterization:

• Conduct co-immunoprecipitation experiments with tagged versions of both proteins

• Perform proximity ligation assays to visualize interactions in situ

• Use yeast two-hybrid or mammalian two-hybrid systems for domain mapping

• Employ FRET/BRET approaches to monitor interaction dynamics in living cells

Structural Studies:

• Express and purify the interacting domains of both proteins

• Perform X-ray crystallography or NMR spectroscopy to determine the structure of the complex

• Use computational modeling to predict interaction interfaces

• Validate structural predictions with site-directed mutagenesis

Functional Consequences:

• Analyze the impact of PDCD4 on RPS13's ribosomal functions

• Investigate how RPS13 affects PDCD4's known tumor suppressor activities

• Examine the influence of this interaction on translation regulation

• Study effects on apoptosis pathways, connecting to RPS13's reported role in drug resistance

Regulatory Mechanisms:

• Determine if post-translational modifications regulate the interaction

• Investigate whether cellular stress conditions modulate the binding

• Examine cell-cycle dependence of the interaction

• Explore tissue-specific variations in the interaction pattern

When designing experiments, consider that PDCD4 is known to interact with translation initiation factors (particularly eIF4A), which may provide context for understanding how RPS13-PDCD4 interaction influences translation regulation beyond canonical ribosomal functions.

How can I address common challenges in RPS13 expression and purification?

Researchers frequently encounter specific challenges when working with RPS13. Here are methodological solutions to common problems:

Expression Challenges:

Purification Challenges:

• Prevent aggregation by including mild detergents or higher salt concentrations in buffers

• Add reducing agents (like DTT or β-mercaptoethanol) to prevent disulfide bond formation

• Include glycerol (50%) to stabilize the protein during storage

• Use staged purification with multiple chromatographic methods for higher purity

Quality Control Recommendations:

• Verify protein identity by mass spectrometry or N-terminal sequencing

• Assess functional activity through RNA binding assays

• Check for proper folding using circular dichroism

• Analyze aggregation state using size-exclusion chromatography

Storage Optimization:

• Store small aliquots to avoid repeated freeze-thaw cycles

• Consider lyophilization for long-term storage

• Test protein activity after storage to ensure functionality is maintained

• Add carrier proteins for dilute solutions to prevent adsorption to tubes

What are the best approaches to resolve contradictory data in RPS13 functional studies?

When faced with contradictory results in RPS13 research, apply these methodological approaches:

Systematic Analysis of Variables:

• Evaluate cellular context differences (cell types, growth conditions)

• Compare protein expression levels across studies (physiological vs. overexpression)

• Assess methodology differences (in vitro vs. in vivo, acute vs. chronic manipulation)

• Review genetic background of model systems used

Technical Validation:

• Repeat experiments with multiple methodologies (e.g., different knockdown techniques)

• Use complementary approaches to measure the same outcome

• Verify antibody specificity with appropriate controls

• Include rescue experiments to confirm specificity of observed phenotypes

Reconciliation Strategies:

• Develop unifying hypotheses that explain seemingly contradictory results

• Consider context-dependent regulation or functions

• Investigate potential post-translational modifications that might explain functional differences

• Examine potential compensatory mechanisms in different experimental systems

Experimental Design for Resolution:

• Design decisive experiments specifically targeting the contradiction

• Include side-by-side comparisons of conflicting methodologies

• Collaborate with labs reporting contradictory results

• Implement blinded analysis to reduce unconscious bias

When publishing results, transparently discuss contradictions in the literature and provide evidence supporting your interpretation while acknowledging alternative explanations.

How can I distinguish between RPS13's ribosomal and extraribosomal functions experimentally?

Differentiating between canonical ribosomal functions and extraribosomal roles of RPS13 requires careful experimental design:

Strategic Mutant Design:

• Create RPS13 variants with mutations that specifically disrupt ribosome incorporation

• Develop mutations that maintain ribosomal integration but alter putative extraribosomal functions

• Use structure-guided design based on available ribosome structural data

• Test each mutant for ribosomal incorporation before functional studies

Differential Knockdown/Rescue Approaches:

• Deplete endogenous RPS13 using siRNA targeting UTRs

• Rescue with wild-type or specific mutant versions

• Compare phenotypic outcomes between different rescue constructs

• Analyze both ribosomal (translation) and non-ribosomal (e.g., apoptosis) readouts

Subcellular Localization Studies:

• Use immunofluorescence or fluorescent protein fusions to track RPS13 localization

• Compare distribution to established ribosomal markers

• Identify non-ribosomal pools of RPS13 through co-localization studies

• Implement live-cell imaging to track dynamic localization changes

Biochemical Fractionation:

• Separate ribosomal and non-ribosomal cellular fractions

• Quantify RPS13 distribution across fractions

• Identify unique interaction partners in different fractions

• Compare stress-induced changes in distribution

When analyzing data, consider that changes in RPS13 will inherently affect ribosome biogenesis and function, potentially causing indirect effects that may be misinterpreted as direct extraribosomal functions.

What emerging technologies could advance our understanding of RPS13 biology?

Several cutting-edge technologies hold promise for deepening our understanding of RPS13 functions:

CRISPR-Based Technologies:

• CRISPRi/CRISPRa for precise modulation of RPS13 expression

• Base editing for introduction of specific mutations without double-strand breaks

• Prime editing for precise nucleotide changes in the RPS13 gene

• CRISPR screening to identify genetic interactors of RPS13

Advanced Imaging Approaches:

• Super-resolution microscopy to visualize RPS13 within ribosomal complexes

• Live-cell imaging with split fluorescent proteins to monitor dynamic interactions

• APEX2 proximity labeling to map the RPS13 neighborhood in living cells

• Correlative light and electron microscopy for ultrastructural localization

Transcriptome/Proteome Analysis:

• Ribosome profiling to identify RPS13-dependent translation events

• Targeted RNA-seq of ribosome-associated mRNAs

• Proteomics to identify RPS13-dependent changes in protein expression

• Proximity-dependent biotin identification (BioID) to map protein interaction networks

Structural Biology Innovations:

• Cryo-electron tomography to visualize RPS13 in cellular context

• Integrative structural biology combining multiple data sources

• Time-resolved structural studies to capture dynamic conformational changes

• AlphaFold2 and related AI approaches to predict interaction interfaces

Each of these technologies offers unique advantages for specific aspects of RPS13 research, and often the combination of multiple approaches provides the most comprehensive understanding.

How might understanding RPS13 autoregulation inform therapeutic strategies?

The autoregulatory mechanism of RPS13, whereby the protein inhibits splicing of its own pre-mRNA , has potential therapeutic implications that could be explored through these research directions:

Therapeutic Target Evaluation:

• Assess whether disruption of RPS13 autoregulation affects cancer cell survival

• Investigate potential selectivity between normal and malignant cells

• Determine if RPS13 autoregulation interfaces with drug resistance mechanisms

• Explore connections to other splicing-related therapeutic targets

Drug Discovery Approaches:

• Screen for small molecules that modulate RPS13-pre-mRNA interaction

• Develop antisense oligonucleotides targeting regulatory elements in intron 1

• Design RNA decoys to sequester excess RPS13 and prevent autoregulation

• Identify natural products that might interfere with RPS13 splicing regulation

Mechanistic Considerations for Therapy Development:

• Map the precise binding site of RPS13 on its pre-mRNA for targeted intervention

• Characterize structural features of the RPS13-RNA complex

• Identify cofactors involved in the autoregulatory process

• Investigate tissue-specific variations in autoregulation that might affect therapeutic window

Translational Research Roadmap:

• Validate targets in patient-derived samples

• Develop appropriate biomarkers for patient stratification

• Design rational combination strategies based on molecular mechanisms

• Establish appropriate preclinical models for testing therapeutic hypotheses

When considering the therapeutic potential, researchers should carefully balance potential benefits against the fundamental importance of RPS13 in protein synthesis, which could limit the therapeutic window of any intervention targeting this pathway.

What are the most promising directions for understanding RPS13's role in cancer and other diseases?

Based on current knowledge and emerging trends, these research directions hold significant promise for elucidating RPS13's role in disease:

Cancer Biology:

• Investigate RPS13 expression patterns across cancer types and correlation with patient outcomes

• Explore mechanistic connections between RPS13 and drug resistance pathways

• Examine potential interactions with cancer-associated signaling networks

• Assess RPS13's influence on cancer-specific translation programs

Ribosomopathies:

• Evaluate whether RPS13 mutations or dysregulation contribute to known ribosomopathies

• Investigate tissue-specific manifestations of RPS13 dysfunction

• Explore connections between RPS13 and ribosomal stress responses

• Develop models to study RPS13-associated developmental abnormalities

Immunological Contexts:

• Examine RPS13's potential role in immune cell translation regulation

• Investigate connections to inflammatory responses

• Explore potential autoimmune aspects related to RPS13 (e.g., as an autoantigen)

• Assess implications for immune surveillance in cancer

Neurodegenerative Diseases:

• Study RPS13's role in neuron-specific translation regulation

• Investigate potential connections to protein aggregation disorders

• Examine stress granule dynamics in relation to RPS13 function

• Assess implications for age-related translation dysregulation

For maximum impact, researchers should consider integrating these disease-focused studies with basic mechanistic investigations of RPS13 function, employing interdisciplinary approaches that combine molecular, cellular, and physiological perspectives.