Recombinant Saccharomyces exiguus 60S ribosomal protein L13 (RPL13)

What is the structure and function of RPL13 in Saccharomyces exiguus?

RPL13 is a component of the 60S subunit of ribosomes, which together with the 40S subunit forms the complete ribosomal complex. The 60S subunit contains approximately 80 structurally distinct proteins along with 4 RNA species that collectively catalyze protein synthesis . RPL13 belongs to the L13E family of ribosomal proteins and is primarily located in the cytoplasm .

How is recombinant Saccharomyces exiguus RPL13 typically expressed?

Recombinant RPL13 from Saccharomyces exiguus can be expressed through several systems, with cell-free protein synthesis (CFPS) being a notable approach. The ALiCE® system, derived from Nicotiana tabacum c.v. lysate, provides the complete protein expression machinery needed for even difficult-to-express proteins requiring post-translational modifications .

For expression, the cDNA encoding the full RPL13 sequence is typically cloned into an appropriate expression vector. For example, the cDNA can be digested with specific restriction enzymes such as EcoRI and XhoI and inserted into a vector like pCMV-N-FLAG to obtain FLAG-tagged RPL13 . The complete amino acid sequence of RPL13 must be confirmed to ensure proper expression of the functional protein .

What methods are used to purify recombinant RPL13?

Purification of recombinant RPL13 typically employs affinity chromatography using tags such as the Strep tag, which facilitates one-step purification with high specificity . The purification process generally follows these steps:

• Expression of the tagged recombinant protein in the chosen system

• Cell lysis or collection of the cell-free expression product

• Binding of the tagged protein to an affinity resin

• Washing steps to remove contaminants

• Elution of the purified protein using competition with free ligand or other elution methods

• Quality control analysis via SDS-PAGE and Western blotting to confirm purity and identity

This approach yields purified recombinant RPL13 suitable for downstream applications including structural studies, functional assays, and interaction analyses.

What extraribosomal functions has RPL13 been shown to perform?

Beyond its canonical role in ribosome structure and protein translation, RPL13 demonstrates significant extraribosomal functions, particularly in immune response regulation:

• Antiviral activity: RPL13 participates in antiviral immune responses and can inhibit viral replication, specifically demonstrated against foot-and-mouth disease virus (FMDV) .

• Immune signaling: Overexpression of RPL13 promotes the induction and activation of nuclear factor-κB (NF-κB) and interferon-β (IFN-β) gene promoters .

• Cytokine regulation: RPL13 enhances the expression and protein secretion of the antiviral factor IFN-β and the proinflammatory cytokine interleukin-6 (IL-6) .

• Translational control: RPL13A (a related protein) can participate in the formation of complexes like the GAIT (gamma interferon-activated inhibitor of translation) complex, which controls inflammation by inhibiting translation of specific inflammatory genes .

These multifaceted functions highlight the evolving understanding of ribosomal proteins as multifunctional entities beyond their structural roles in ribosomes.

How does RPL13 interact with viral components during infection?

Research has revealed specific interactions between RPL13 and viral components:

• FMDV 3C protease interaction: The 3C protease of foot-and-mouth disease virus directly interacts with RPL13. This interaction appears to be a viral evasion mechanism, as the protease activity reduces RPL13 expression, thereby antagonizing the RPL13-mediated antiviral activity .

• Complex formation: In response to respiratory syncytial virus (RSV) infection, RPL13A can be released from the 60S large subunit and recognize specific hairpin structures in viral mRNA. This leads to the formation of a virus-activated translation inhibition complex (VAIT) that inhibits viral protein translation .

• Signaling pathway modulation: RPL13 appears to influence antiviral signaling cascades, potentially through interactions with pathway components that affect the cellular response to viral infection .

These interactions demonstrate the sophisticated role RPL13 plays in host-pathogen dynamics and suggest potential therapeutic applications targeting these interactions.

What experimental approaches are used to study RPL13's role in ribosome assembly?

Studying RPL13's role in ribosome assembly requires sophisticated experimental approaches:

• Pre-rRNA processing analysis: Researchers examine the processing of 27SA3 pre-rRNA to 27SB1S pre-rRNA, a critical step in 60S ribosomal subunit assembly that can be affected by RPL13 and associated assembly factors .

• Assembly factor interdependence studies: Techniques to deplete specific assembly factors and analyze their effects on ribosome assembly help establish the sequential and interdependent nature of assembly components .

• Protein-RNA interaction mapping: Methods to identify binding sites of RPL13 on ribosomal RNA provide insights into the structural role of the protein within the ribosome complex .

• Neighborhood analysis: Examining interactions with nearby ribosomal proteins (such as rpL17, rpL26, rpL35, and rpL37 in S. cerevisiae) reveals functional neighborhoods within the ribosome that depend on proper RPL13 assembly .

These approaches collectively provide a comprehensive understanding of RPL13's role in the complex process of ribosome biogenesis.

How can researchers assess the functional activity of recombinant RPL13?

Assessing RPL13 functional activity involves several methodological approaches:

Table 1: Methods for Assessing RPL13 Functional Activity

For functional studies, researchers typically employ overexpression and knockdown approaches. Overexpression of RPL13 can be achieved using recombinant expression plasmids, while knockdown is commonly performed using RNA interference techniques or CRISPR-Cas9 gene editing . The functional consequences are then assessed using the methods described in the table above.

What are the current challenges in expressing and purifying active recombinant RPL13?

Despite advances in recombinant protein expression, several challenges remain in producing active RPL13:

• Structural complexity: As a ribosomal protein, RPL13 normally exists within the complex environment of the ribosome, potentially requiring specific conditions or chaperones for proper folding when expressed recombinantly .

• Solubility issues: Recombinant expression can lead to inclusion body formation, necessitating optimization of expression conditions or refolding protocols to obtain soluble protein .

• Post-translational modifications: If RPL13 requires specific post-translational modifications for activity, expression systems must be selected that can perform these modifications correctly .

• Functional assessment: Determining whether recombinantly expressed RPL13 retains all its native functions, especially extraribosomal ones, presents analytical challenges .

• Expression system selection: Different expression systems (bacterial, yeast, insect, mammalian, cell-free) offer different advantages and limitations for RPL13 expression, requiring careful consideration based on downstream applications .

The ALiCE® cell-free protein synthesis system has shown promise in addressing some of these challenges by providing the necessary machinery for proper protein folding and potential post-translational modifications .

How can protein-protein interactions of RPL13 be studied effectively?

Understanding RPL13's interactions with other proteins is crucial for elucidating its functions. Several methodologies can be employed:

• Co-immunoprecipitation (Co-IP): Using antibodies against RPL13 or its interaction partners to pull down protein complexes, followed by Western blotting or mass spectrometry to identify interacting proteins. This approach has identified interactions such as RPL13 with CDC5L and FMDV 3C protease .

• Yeast two-hybrid screening: A genetic approach to detect protein-protein interactions that can identify novel binding partners of RPL13.

• Proximity labeling: Methods like BioID or APEX can identify proteins in close proximity to RPL13 within cells, providing insights into its interaction network.

• Structural studies: X-ray crystallography or cryo-electron microscopy of RPL13 in complex with interaction partners can reveal the molecular details of these interactions.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These techniques provide quantitative measurements of binding affinities between RPL13 and its partners.

These complementary approaches provide a comprehensive understanding of RPL13's interactome and the functional significance of these interactions.

How is RPL13 involved in disease processes beyond viral infections?

RPL13's involvement in disease extends beyond viral infections to several pathological conditions:

Research into these areas is ongoing, with emerging evidence supporting RPL13's multifaceted roles in health and disease beyond its canonical ribosomal function.

What technological advances have improved our understanding of RPL13 structure and function?

Recent technological developments have significantly enhanced our understanding of RPL13:

• Cryo-electron microscopy: High-resolution structures of ribosomes have revealed the precise positioning of RPL13 within the ribosomal complex and its interactions with ribosomal RNA and neighboring proteins .

• CRISPR-Cas9 gene editing: This technology has enabled precise manipulation of RPL13 expression and sequence in various cellular contexts, facilitating functional studies with unprecedented specificity.

• Ribosome profiling: This technique provides genome-wide information on ribosome positioning on mRNAs, helping to elucidate how alterations in RPL13 affect translation globally.

• Cell-free expression systems: Advanced systems like ALiCE® have improved the production of recombinant RPL13 for structural and functional studies .

• Proteomics approaches: Mass spectrometry-based techniques have enabled comprehensive analysis of RPL13's post-translational modifications and interaction partners under various conditions.

These technological advances continue to expand our understanding of RPL13's multifaceted roles in cellular processes.

What are the implications of RPL13's extraribosomal functions for therapeutic development?

The discovery of RPL13's extraribosomal functions opens new avenues for therapeutic development:

• Antiviral strategies: Understanding how RPL13 contributes to antiviral immunity and how viruses counteract this activity could inform the development of novel antiviral therapies. For example, preventing the interaction between FMDV 3C protease and RPL13 might enhance natural antiviral defenses .

• Immunomodulatory approaches: RPL13's role in regulating inflammatory cytokines suggests potential applications in controlling excessive inflammation in conditions like autoimmune diseases or sepsis.

• Cancer therapeutics: The differential expression of RPL13 in benign versus malignant breast tissue suggests potential diagnostic or therapeutic applications in cancer .

• Targeted protein degradation: Novel approaches like PROTACs (proteolysis targeting chimeras) could potentially be designed to selectively modulate RPL13 levels or functions in specific disease contexts.

• Structure-based drug design: Detailed structural information about RPL13 and its interaction partners could facilitate the design of small molecules that selectively modulate these interactions.

These potential therapeutic applications highlight the importance of continued research into RPL13's diverse functions.