Recombinant Schizosaccharomyces pombe 40S ribosomal protein S13 (rps13)

What is the structural characterization of S. pombe rps13?

S. pombe rps13 (UniProt ID: P28189, also known as RS13_SCHPO) is a Small ribosomal subunit protein uS15 encoded by the gene rps13 (ORF name: SPAC6F6.07c) in Schizosaccharomyces pombe strain 972. This protein functions as a component of the 40S ribosomal subunit and belongs to the universal ribosomal protein uS15 family . As part of the small ribosomal subunit, rps13 plays a critical role in the ribonucleoprotein complex responsible for protein synthesis in the cell . The protein participates in ribosome assembly and contributes to the structural integrity of the 40S subunit.

To determine the structural properties of S. pombe rps13, researchers typically employ X-ray crystallography or cryo-electron microscopy techniques that reveal its three-dimensional conformation within the ribosomal complex. These methods help elucidate how rps13 interacts with ribosomal RNA and neighboring proteins to maintain the functional architecture of the small subunit. Additionally, sequence analysis and structural prediction can provide insights into conserved domains and functional motifs that are essential for its role in translation.

What post-translational modifications are documented for S. pombe rps13?

S. pombe rps13 undergoes multiple post-translational modifications that likely regulate its function and interactions. According to the iPTMnet database, the protein exhibits a complex pattern of modifications including phosphorylation, ubiquitination, and sumoylation at various sites . These modifications may affect ribosome assembly, translation efficiency, or extraribosomal functions of rps13.

The documented post-translational modifications include:

To experimentally verify these modifications, researchers typically employ mass spectrometry-based proteomics approaches. Phosphorylation sites can be identified using phospho-enrichment followed by LC-MS/MS analysis, while ubiquitin remnant profiling can detect ubiquitinated lysine residues. Understanding the dynamics and functional consequences of these modifications requires additional biochemical and genetic approaches.

How is S. pombe rps13 involved in ribosome biogenesis?

S. pombe rps13 plays a crucial role in ribosome biogenesis as a component of the small subunit (SSU) processome, which is the first precursor of the small eukaryotic ribosomal subunit . During the assembly of the SSU processome in the nucleolus, ribosome biogenesis factors, RNA chaperones, and ribosomal proteins like rps13 associate with the nascent pre-rRNA. These components work together to facilitate RNA folding, modifications, rearrangements, and cleavage events .

The protein contributes to the processing of pre-ribosomal RNA and the structural organization of the 40S subunit. Research indicates that rps13 associates with specific rRNA regions, including the 5.8S rRNA , suggesting a role in stabilizing RNA-protein interactions during ribosome assembly. Additionally, rps13 may influence the targeted degradation of pre-ribosomal RNA by the RNA exosome, a process that ensures quality control during ribosome biogenesis .

To study rps13's role in ribosome biogenesis, researchers can use genetic approaches to create conditional mutants, followed by analysis of pre-rRNA processing intermediates. Northern blotting, qRT-PCR, and pulse-chase labeling of rRNA provide insights into how rps13 dysfunction affects specific stages of ribosome assembly.

What are the optimal expression systems for producing recombinant S. pombe rps13?

Several expression systems can be employed for the production of recombinant S. pombe rps13, each with distinct advantages depending on research objectives:

• Escherichia coli expression system: This represents a widely used platform for recombinant protein production due to its rapid growth, high yield, and established protocols. Similar to human RPS13 expression mentioned in the literature , E. coli can be utilized for S. pombe rps13 expression with appropriate codon optimization. This system is particularly suitable when large quantities of protein are needed for structural studies or when post-translational modifications are not essential for the research question.

• S. pombe homologous expression: Using S. pombe itself as an expression host offers significant advantages when studying a protein native to this organism. S. pombe strain Q01 has been reported as a good transformation host and expression system . This approach ensures proper folding and post-translational modifications that would occur naturally, making it ideal for functional studies where physiological relevance is paramount.

• Integrative vector systems: For stable expression in S. pombe, integrative vectors like pYIplac128 provide consistent expression levels by incorporating the recombinant gene into the chromosome . This approach circumvents problems associated with plasmid loss during fermentation, which can significantly impact product yield .

The methodology for expressing recombinant S. pombe rps13 typically involves:

• Cloning the rps13 gene into an appropriate expression vector with suitable regulatory elements

• Transformation of the construct into the chosen host organism

• Optimization of culture conditions for protein expression

• Protein purification using affinity chromatography or other separation techniques

For challenging expression projects, optimizing parameters such as temperature, inducer concentration, and co-expression with chaperones may significantly improve yield and solubility.

How can synthetic genetic array (SGA) techniques be applied to study rps13 function in S. pombe?

Synthetic genetic array (SGA) analysis provides a powerful approach for studying gene function through systematic analysis of genetic interactions. This technique has been successfully adapted for S. pombe, offering valuable insights into gene functions and relationships . For studying rps13 function, SGA can be applied through the following methodology:

• Generation of rps13 query strains: As described in the literature, query strains can be created by introducing modifications to the rps13 gene along with a selectable marker (e.g., NatMX cassette) . These modifications might include complete gene deletion (if not lethal), domain deletions, or point mutations that alter specific functions. The modifications are typically introduced through homologous recombination by transforming S. pombe with a PCR product containing the marker and homologous flanking regions .

• Strain crossing and selection: The rps13 query strain is systematically crossed with an array of deletion or mutation strains covering the S. pombe genome. Through a series of selection steps on appropriate media, haploid double mutants containing both the rps13 modification and a second gene alteration are isolated.

• Phenotypic analysis and interaction scoring: Growth rates or other phenotypic characteristics of the double mutants are compared to the single mutants to identify genetic interactions. Interactions are typically classified as synthetic lethal, synthetic sick (negative interactions), or suppressive (positive interactions).

• Data analysis and network construction: The pattern of genetic interactions forms a network that can reveal functional relationships between rps13 and other genes. Genes with similar interaction profiles often function in related pathways.

S. pombe offers distinct advantages for SGA studies compared to S. cerevisiae, including less functional redundancy in its genome, which leads to cleaner phenotypic consequences and easier interpretation of results . This approach can reveal both expected interactions with other ribosomal components and potentially novel connections to other cellular processes.

What methodologies can determine the RNA binding specificity of S. pombe rps13?

Understanding the RNA binding specificity of rps13 is crucial for elucidating its role in ribosome assembly and function. Several complementary techniques can be employed to comprehensively characterize these interactions:

• CLIP-seq (Cross-linking and immunoprecipitation followed by sequencing): This technique involves UV cross-linking of protein-RNA complexes in vivo, followed by immunoprecipitation of rps13 (using either antibodies against the native protein or an epitope tag) and sequencing of the bound RNA fragments. CLIP-seq provides a genome-wide view of rps13-RNA interactions in their cellular context. Variations include PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced CLIP) and iCLIP (individual-nucleotide resolution CLIP), each with specific advantages for resolution and quantification.

• RNA Electrophoretic Mobility Shift Assay (EMSA): For in vitro validation of specific interactions, purified recombinant rps13 protein can be incubated with labeled RNA fragments of interest. Formation of protein-RNA complexes is detected as a mobility shift on a native gel. This approach allows determination of binding affinity and specificity for different RNA sequences.

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI): These techniques enable real-time analysis of binding kinetics between rps13 and various RNA substrates, providing quantitative measurements of association and dissociation rates as well as binding affinities.

• RNA footprinting: Chemical or enzymatic probing of RNA structure in the presence and absence of rps13 can identify regions protected by protein binding. Methods such as SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or DMS-MaPseq (Dimethyl sulfate Mutational Profiling with sequencing) provide nucleotide-resolution information about RNA structure changes upon protein binding.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach can identify regions of rps13 that undergo conformational changes upon RNA binding, providing insights into the structural basis of the interaction.

By integrating data from these complementary approaches, researchers can build a comprehensive model of rps13-RNA interactions, identifying both the RNA sequences recognized by the protein and the structural elements of rps13 involved in binding.

How can I design experiments to study the effect of rps13 phosphorylation on ribosome function?

Investigating the functional consequences of rps13 phosphorylation requires a multi-faceted experimental approach that combines genetic, biochemical, and functional assays. Based on the documented phosphorylation sites in S. pombe rps13 (S13, S14, Y18, S21, S32, S46, S48, Y89) , the following experimental design can be implemented:

• Generation of phosphomutant strains: Create S. pombe strains expressing rps13 variants with mutations at specific phosphorylation sites:

  • Phosphodeficient mutants: Replace serine/threonine/tyrosine residues with alanine to prevent phosphorylation

  • Phosphomimetic mutants: Replace serine/threonine with glutamate or aspartate to mimic constitutive phosphorylation

  • Single-site mutants to study individual sites and combinatorial mutants to investigate potential cross-talk between sites

  These mutants can be created using site-directed mutagenesis and integrated into the S. pombe genome using homologous recombination techniques as described in the literature .

• Phenotypic characterization: Assess the impact of phosphomutants on:

  • Cell growth under various conditions (temperature, nutrients, stress)

  • Cell cycle progression

  • Sensitivity to translation inhibitors

  • Global protein synthesis rates using metabolic labeling

• Ribosome profiling: Apply ribosome profiling to compare translational efficiency and ribosome positioning in wild-type and phosphomutant strains. This technique provides genome-wide information about translation dynamics and can reveal changes in start site selection, elongation rates, or ribosome stalling.

• Polysome analysis: Use sucrose gradient centrifugation to separate and quantify different ribosomal species (40S, 60S, 80S, and polysomes). Alterations in the polysome profile can indicate defects in subunit joining, translation initiation, or elongation.

• In vitro translation assays: Purify ribosomes from wild-type and phosphomutant strains and compare their activity in translating reporter mRNAs in a reconstituted translation system.

• Structural analysis: Apply cryo-electron microscopy to determine if phosphorylation or its absence affects the structure of the 40S subunit or the conformation of rps13 within the ribosome.

• Identification of phosphorylation-dependent interactions: Use affinity purification coupled with mass spectrometry to identify proteins that differentially interact with wild-type versus phosphomutant rps13, potentially revealing phosphorylation-regulated interactions.

This comprehensive approach can establish causal relationships between specific phosphorylation events and functional outcomes, providing insights into how post-translational modifications regulate ribosome function in S. pombe.

What controls should be included when using S. pombe as an expression system for recombinant rps13?

When using S. pombe as an expression system for recombinant rps13, appropriate controls are essential to ensure the validity and interpretability of results. The following controls should be considered:

• Vector-only control: Cells transformed with the expression vector lacking the rps13 insert help distinguish effects caused by the vector itself from those due to rps13 expression.

• Wild-type endogenous rps13 control: Unmodified S. pombe expressing only the endogenous rps13 provides a baseline for comparing expression levels, post-translational modifications, and functional characteristics of the recombinant protein.

• Tagged endogenous rps13 control: If the recombinant rps13 includes tags (e.g., His-tag, FLAG-tag), creating a strain with an identically tagged endogenous rps13 helps assess the impact of the tag on protein function.

• Expression level controls:

  • Strains with varying expression levels of recombinant rps13 (achieved through different promoters or copy numbers) help establish dose-dependent effects

  • Inducible expression systems allow controlled comparison of the same cells before and after rps13 induction

• Integration site controls: If using integrative vectors like pYIplac128 , control strains with integration at different genomic locations help distinguish positional effects from genuine rps13-related phenotypes.

• Post-translational modification controls:

  • Phosphatase treatment of cell extracts to remove phosphorylation

  • Inhibitors of specific modification pathways to assess their contribution to rps13 function

  • Comparison with E. coli-expressed rps13 lacking eukaryotic modifications

• Functional rescue controls: In strains where endogenous rps13 is depleted or inactivated, testing whether recombinant variants can rescue the phenotype provides evidence for functional equivalence or deficiency.

• Strain background controls: Expressing recombinant rps13 in different S. pombe strain backgrounds helps ensure that observed effects are not strain-specific.

By systematically incorporating these controls, researchers can confidently attribute observed phenotypes to specific properties of the recombinant rps13 protein, minimizing the risk of misinterpreting artifacts of the expression system.

How can I investigate interactions between rps13 and other components of the ribosome assembly pathway?

Investigating interactions between rps13 and other components of the ribosome assembly pathway requires a combination of genetic, biochemical, and imaging approaches. The following experimental strategies provide a comprehensive framework:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express epitope-tagged rps13 in S. pombe

  • Perform immunoprecipitation under conditions that preserve native complexes

  • Identify co-purifying proteins by mass spectrometry

  • Use staged purifications at different timepoints during ribosome biogenesis to capture dynamic interactions

  • Compare interaction profiles between wild-type rps13 and mutant variants to identify functionally important interactions

• Proximity labeling approaches:

  • Express rps13 fused to a proximity labeling enzyme (BioID, TurboID, or APEX2)

  • Allow biotinylation of proteins in close proximity to rps13 in living cells

  • Purify biotinylated proteins and identify them by mass spectrometry

  • This approach captures transient interactions that might be missed by conventional AP-MS

• Yeast two-hybrid (Y2H) screening:

  • Use rps13 as bait to screen for interacting proteins

  • Perform directed Y2H assays to test interactions with specific ribosome assembly factors

  • Validate positive interactions with alternative methods

• Genetic interaction analysis:

  • Apply synthetic genetic array (SGA) methodology as described earlier

  • Focus on genetic interactions with known ribosome assembly factors

  • Identify suppressor or enhancer relationships that suggest functional connections

• Co-localization studies:

  • Use fluorescence microscopy with differentially tagged rps13 and ribosome assembly factors

  • Apply techniques like FRET (Förster Resonance Energy Transfer) to detect direct interactions

  • Perform time-lapse imaging to track the dynamics of interactions during ribosome assembly

• RNA-protein interaction analysis:

  • Use CLIP-seq to identify RNAs bound by rps13

  • Compare rps13 binding sites with those of other ribosome assembly factors

  • Investigate how mutations in rps13 affect RNA binding by other factors and vice versa

• In vitro reconstitution:

  • Purify recombinant rps13 and selected assembly factors

  • Reconstitute subcomponents of the assembly pathway in vitro

  • Use biochemical assays to measure the impact of rps13 on the activities of assembly factors

How can I troubleshoot issues with recombinant rps13 solubility and stability?

Ribosomal proteins like rps13 often present challenges in recombinant expression due to their natural incorporation into large ribonucleoprotein complexes. The following troubleshooting strategies address common issues with solubility and stability:

• Expression system optimization:

  • If using E. coli, try specialized strains designed for expressing difficult proteins (e.g., BL21(DE3)pLysS, Rosetta, or Arctic Express)

  • Consider switching to S. pombe expression for native folding environments

  • Test different induction temperatures (16-30°C), with lower temperatures generally favoring proper folding

  • Use a range of inducer concentrations to find the optimal expression level that balances yield with solubility

• Fusion partners and solubility tags:

  • Test N-terminal fusion tags like MBP (maltose-binding protein), GST (glutathione S-transferase), or SUMO, which can significantly enhance solubility

  • Compare with C-terminal tags, as tag position can dramatically affect folding

  • Consider dual tagging strategies for both purification and solubility enhancement

  • Include appropriate linker sequences between rps13 and fusion partners

• Buffer optimization:

  • Screen different pH values (typically 6.0-8.5) to identify the optimal range for stability

  • Test various salt concentrations (50-500 mM NaCl) to minimize aggregation

  • Include stabilizing additives: glycerol (5-20%), arginine (50-100 mM), or non-detergent sulfobetaines

  • For purified protein, optimize storage conditions using differential scanning fluorimetry

• Co-expression strategies:

  • Co-express rps13 with interacting partners from the 40S subunit

  • Include ribosomal RNA fragments that interact with rps13

  • Co-express with molecular chaperones to assist proper folding

• Extraction and purification optimization:

  • Use mild cell lysis methods to prevent aggregation during extraction

  • Test different detergents at low concentrations for membrane-associated fractions

  • Implement step gradients rather than harsh elution conditions during purification

  • Consider on-column refolding for proteins recovered from inclusion bodies

• Stability assessment and enhancement:

  • Monitor protein stability over time using activity assays or light scattering

  • Identify and mutate oxidation-prone methionine residues if they affect stability

  • Use nano-differential scanning fluorimetry to identify stabilizing buffer conditions

  • Consider chemical crosslinking to stabilize native conformations

By systematically addressing these aspects, researchers can overcome common obstacles in working with recombinant rps13 and develop protocols that yield stable, functional protein for downstream applications.

What approaches can identify phenotypes associated with specific rps13 post-translational modifications?

Linking specific post-translational modifications (PTMs) of rps13 to phenotypic outcomes requires a multi-faceted approach that combines genetic, biochemical, and systems-level analyses:

• Site-specific mutant analysis:

  • Generate a panel of S. pombe strains expressing rps13 variants with mutations at specific PTM sites

  • Create both non-modifiable mutants (e.g., S→A for phosphorylation sites, K→R for ubiquitination/sumoylation sites) and modification-mimicking mutants (e.g., S→E for phosphorylation)

  • Perform comprehensive phenotypic characterization including:

    • Growth rate analysis under various conditions

    • Cell cycle progression

    • Response to various stressors (oxidative, heat, osmotic)

    • Sensitivity to translation inhibitors

    • Global and specific protein synthesis rates

• Modification-specific interactome analysis:

  • Isolate rps13 under conditions that preserve specific modifications

  • Identify proteins that differentially interact with modified versus unmodified rps13

  • Map these interactions to specific cellular pathways that might be affected

• Global impact assessment:

  • Compare transcriptome and proteome profiles between wild-type and PTM-mutant strains

  • Perform ribosome profiling to identify changes in translation efficiency

  • Use polysome profiling to assess effects on ribosome assembly and translation

  • Apply statistical approaches to identify significantly altered genes or pathways

• Dynamic modification analysis:

  • Monitor changes in specific rps13 modifications in response to different conditions or stresses

  • Correlate modification changes with cellular phenotypes

  • Identify the enzymes responsible for adding and removing modifications (kinases/phosphatases, E3 ligases/deubiquitinases)

  • Generate mutants of these enzymes to confirm their role in rps13 regulation

• Structural and functional studies:

  • Use structural biology approaches to determine how specific modifications affect rps13 conformation

  • Perform in vitro translation assays with ribosomes containing modified or unmodified rps13

  • Assess the impact of modifications on RNA binding and interactions with other ribosomal components

• Systems-level data integration:

  • Apply network analysis tools to integrate data from multiple experiments

  • Identify key pathways and processes affected by specific modifications

  • Use machine learning approaches to predict functional consequences of modifications based on their molecular context

By systematically applying these approaches, researchers can establish causal relationships between specific PTMs of rps13 and their functional consequences, providing insights into how these modifications regulate ribosome function and potentially extraribosomal activities of rps13.

How can I validate the specificity of antibodies for detecting S. pombe rps13 and its modified forms?

Ensuring antibody specificity is crucial for reliable detection of rps13 and its post-translationally modified forms in research applications. The following comprehensive validation strategy addresses this challenge:

• Genetic controls:

  • Test antibody reactivity against samples from wild-type S. pombe and rps13 deletion strains (if viable) or depletion strains

  • Include rps13 overexpression samples to confirm signal intensity correlation with protein levels

  • Validate with point mutants affecting specific recognition epitopes

  • For modification-specific antibodies, test reactivity against samples from strains expressing non-modifiable rps13 mutants (e.g., S→A for phosphorylation sites)

• Biochemical validation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm the identity of captured proteins

  • For modification-specific antibodies, treat samples with enzymes that remove the modification (e.g., phosphatases for phospho-specific antibodies) and confirm signal loss

  • Compare reactivity against recombinant rps13 expressed in E. coli (unmodified) versus S. pombe (naturally modified)

  • Perform peptide competition assays using the immunizing peptide to demonstrate specificity

• Cross-reactivity assessment:

  • Test antibody against closely related proteins, particularly other ribosomal proteins

  • Evaluate potential cross-reactivity with human or other model organism homologs if working in heterologous systems

  • For modification-specific antibodies, test cross-reactivity with the same modification on different proteins

• Technical validation across methods:

  • Compare results across multiple techniques (Western blot, immunofluorescence, immunoprecipitation)

  • Verify size specificity using SDS-PAGE with appropriate molecular weight markers

  • Confirm subcellular localization patterns align with known rps13 distribution

  • Test multiple antibody lots to ensure consistency

• Quantitative validation:

  • Establish the linear detection range for quantitative applications

  • Determine limits of detection and quantification

  • Validate reproducibility across technical and biological replicates

  • Compare results with orthogonal detection methods (e.g., targeted mass spectrometry)

• Modification-specific validation:

  • For phospho-specific antibodies, confirm signal increases after treatment with phosphatase inhibitors or decreases after phosphatase treatment

  • For ubiquitin/SUMO-specific detection, verify signal enhancement after proteasome inhibition

  • Use in vitro modification systems to generate positive controls with defined modification states

  • Compare antibody-based detection with mass spectrometry-based PTM site mapping

By implementing this comprehensive validation strategy, researchers can ensure high confidence in antibody-based detection of S. pombe rps13 and its modified forms, enabling reliable analysis of this protein in various experimental contexts.

How can CRISPR-Cas9 genome editing be optimized for studying rps13 in S. pombe?

CRISPR-Cas9 technology offers powerful capabilities for precise genetic manipulation of S. pombe rps13. While traditional homologous recombination approaches have been well-established in S. pombe , CRISPR-Cas9 provides advantages in efficiency and precision. The following methodological approach outlines optimization strategies:

• Guide RNA design and selection:

  • Design multiple guide RNAs targeting different regions of the rps13 gene using S. pombe-specific CRISPR design tools

  • Prioritize guide RNAs with high on-target scores and minimal predicted off-target effects

  • Consider targeting conserved functional domains for studying specific protein functions

  • For introduction of point mutations, design guide RNAs that cut near the desired modification site

  • Test guide RNA efficiency using in vitro cleavage assays before cellular applications

• Cas9 expression optimization:

  • Use codon-optimized Cas9 for efficient expression in S. pombe

  • Test both constitutive and inducible expression systems

  • Consider nuclear localization signal optimization for efficient nuclear targeting

  • Compare different promoters to identify the optimal expression level that balances editing efficiency with potential toxicity

• Repair template design:

  • For precise modifications, design donor DNA templates with homology arms of appropriate length (typically 40-60 bp for small changes, longer for complex modifications)

  • Include silent mutations in the PAM site or seed region to prevent re-cutting of edited sequences

  • Incorporate selectable markers for efficient isolation of edited cells, with options for marker removal if needed

  • Consider using synthetic donor DNA to avoid PCR-introduced errors

• Delivery method optimization:

  • Compare transformation efficiencies of different delivery formats:

    • Plasmid-based expression of Cas9 and guide RNA

    • Ribonucleoprotein (RNP) complex delivery

    • Split delivery approaches (e.g., stable Cas9 expression with transient guide RNA delivery)

  • Optimize transformation protocols specifically for CRISPR components, potentially modifying traditional lithium acetate methods

• Editing validation and clone selection:

  • Develop efficient screening strategies combining PCR, restriction digestion, and sequencing

  • Use digital droplet PCR for quantitative assessment of editing efficiency

  • Implement next-generation sequencing for comprehensive off-target analysis

  • Verify phenotypic consequences align with expected outcomes based on the nature of the edit

• Advanced CRISPR applications:

  • Adapt base editing systems for introducing point mutations without double-strand breaks

  • Implement prime editing for precise insertions or small deletions

  • Develop CRISPRi (interference) and CRISPRa (activation) systems for conditional regulation of rps13 expression

  • Consider multiplexed editing for simultaneous modification of rps13 and interacting partners

By systematically optimizing these parameters, researchers can establish efficient CRISPR-Cas9 protocols for precise genetic manipulation of rps13 in S. pombe, enabling sophisticated functional studies of this important ribosomal protein.

How can high-throughput proteomics identify the interactome of S. pombe rps13 under different conditions?

Modern proteomics technologies offer powerful approaches for comprehensively mapping the dynamic interactome of S. pombe rps13. The following methodological framework enables systematic identification of condition-specific protein interactions:

• Sample preparation strategies:

  • Generate S. pombe strains expressing affinity-tagged rps13 (e.g., FLAG, HA, or BioID fusion) under native regulatory control

  • Culture cells under diverse conditions of interest:

    • Different growth phases (log, stationary)

    • Various stress conditions (oxidative, temperature, nutrient deprivation)

    • Treatment with translation inhibitors or ribosome biogenesis disruptors

    • Cell cycle stages (synchronized cultures)

  • Perform gentle cell lysis to preserve native protein complexes

  • Use crosslinking approaches (chemical or UV) to capture transient interactions

• Affinity purification methods:

  • Apply single-step and tandem affinity purification protocols to balance sensitivity with specificity

  • Compare different affinity tags and elution conditions to maximize recovery of genuine interactors

  • Include appropriate controls: untagged strains, non-specific bait proteins, and competitive elution

  • Implement isotope labeling (SILAC) or tandem mass tag (TMT) labeling for quantitative comparison across conditions

• Proximity labeling approaches:

  • Express rps13 fused to enzyme tags (BioID, TurboID, or APEX2) that biotinylate nearby proteins

  • Optimize labeling conditions (time, temperature, biotin concentration) for different experimental goals

  • Purify biotinylated proteins using streptavidin-based affinity capture

  • This approach captures both stable and transient interactions in the native cellular environment

• Mass spectrometry analysis:

  • Employ high-resolution mass spectrometry (e.g., Orbitrap or timsTOF systems) for protein identification

  • Implement data-independent acquisition (DIA) for comprehensive and reproducible detection

  • Use targeted proteomics (PRM or MRM) for validation and precise quantification of selected interactions

  • Apply advanced fragmentation methods (ETD, EThcD) for improved peptide identification and PTM characterization

• Data analysis and interpretation:

  • Apply robust statistical methods to distinguish genuine interactors from background contaminants

  • Use SAINT, CRAPome, or similar tools to score interaction confidence

  • Perform hierarchical clustering to identify condition-specific interaction modules

  • Integrate with existing protein interaction databases and literature to place findings in biological context

  • Apply network analysis to identify key hubs and functional modules within the interactome

• Validation and functional characterization:

  • Confirm selected interactions using orthogonal methods (co-immunoprecipitation, yeast two-hybrid)

  • Employ genetic approaches (e.g., double mutant analysis) to validate functional relationships

  • Visualize interactions in living cells using fluorescence microscopy methods

  • Perform structural studies of key complexes to understand the molecular basis of interactions

This comprehensive approach enables researchers to construct dynamic maps of the S. pombe rps13 interactome, revealing how its protein interactions change across conditions and providing insights into both its canonical ribosomal functions and potential extraribosomal roles.

What bioinformatic approaches can predict functional impacts of rps13 mutations and modifications?

Advanced bioinformatic methodologies provide powerful tools for predicting the functional consequences of mutations and post-translational modifications in S. pombe rps13. The following approaches can guide experimental design and interpretation:

• Structural impact prediction:

  • Generate or utilize existing structural models of S. pombe rps13 within the ribosomal context

  • Apply molecular dynamics simulations to predict how mutations or modifications alter protein conformation

  • Calculate changes in stability using tools like FoldX, Rosetta, or DUET

  • Visualize structural changes and identify potentially disrupted interactions

  • Assess conservation of affected residues across species to infer functional importance

• Machine learning-based functional prediction:

  • Train classifiers on existing mutation/modification datasets to predict functional impacts

  • Apply deep learning approaches that integrate multiple data types (sequence, structure, conservation)

  • Use transfer learning from human RPS13 data where direct S. pombe data is limited

  • Develop S. pombe-specific prediction models incorporating yeast-specific features

  • Validate predictions with experimental data in an iterative refinement process

• Network-based analysis:

  • Construct functional networks integrating protein-protein interactions, genetic interactions, and co-expression data

  • Apply network propagation algorithms to predict the systemic impact of rps13 perturbations

  • Identify potential compensatory mechanisms within the network

  • Predict synthetic lethal or suppressor relationships to guide genetic studies

  • Map modifications to interaction interfaces to predict regulatory effects

• Evolutionary analysis:

  • Perform phylogenetic analysis to identify evolutionarily conserved and variable regions

  • Apply evolutionary coupling analysis to detect co-evolving residues that may be functionally linked

  • Compare modification patterns across species to identify conserved regulatory sites

  • Use ancestral sequence reconstruction to understand the evolutionary trajectory of key residues

  • Apply selection pressure analysis to identify functionally constrained regions

• Integration of multi-omics data:

  • Develop integrative models that combine transcriptomic, proteomic, and translatomic data

  • Apply Bayesian network approaches to infer causal relationships

  • Use dimensionality reduction techniques to identify patterns in complex datasets

  • Perform enrichment analysis to identify affected biological processes and pathways

  • Apply time-series analysis for temporal data to understand dynamic responses

• Specific modification impact prediction:

  • Utilize specialized tools for predicting the impact of particular modifications:

    • PhosphoSitePlus and NetPhos for phosphorylation site prediction and functional analysis

    • UbPred and UbiSite for ubiquitination impact prediction

    • SUMOsp and GPS-SUMO for sumoylation analysis

  • Consider the interplay between different modifications using tools that predict PTM crosstalk

By systematically applying these bioinformatic approaches, researchers can generate testable hypotheses about the functional impacts of mutations and modifications in S. pombe rps13, guiding experimental design and providing a framework for interpreting experimental results in a broader biological context.