Recombinant Arabidopsis thaliana 40S ribosomal protein S18 (RPS18A)

What is the basic structure and function of RPS18A in Arabidopsis thaliana?

RPS18A is a core component of the 40S ribosomal subunit in Arabidopsis thaliana. The gene is located on chromosome 1 and contains 4 exons spanning approximately 1466 bp with a coding sequence (CDS) of 459 bp . The protein belongs to the Ribosomal protein S13/S18 family and is also known as PFL (POINTED FIRST LEAVES) and PFL1 (POINTED FIRST LEAVES 1), suggesting its role in leaf development .

Arabidopsis contains three paralogs of this protein: RPS18A, RPS18B, and RPS18C . While these proteins share high sequence similarity, they may have evolved specialized functions or tissue-specific expression patterns. The maintenance of multiple paralogs suggests functional significance beyond simple redundancy.

How does RPS18A contribute to ribosome biogenesis?

RPS18A likely plays dual roles as both a structural component of the mature ribosome and as a ribosome biogenesis factor (RBF). This pattern has been observed for other ribosomal proteins, such as RPS24, which functions in 18S rRNA maturation in Arabidopsis . During ribosome assembly, RPS18A is incorporated into pre-ribosomal particles in the nucleolus, where it may facilitate specific steps in pre-rRNA processing.

Similar to RPS24, which influences the processing of pre-rRNA intermediates , RPS18A potentially participates in critical cleavage events within the internal transcribed spacer regions (ITS) that are necessary for generating mature 18S rRNA. Studies on RPS24 have shown that deficiencies in ribosomal proteins can lead to accumulation of specific pre-rRNA species, particularly affecting the ITS1-first pathway that contributes significantly to 18S rRNA production .

Methodologically, researchers can investigate RPS18A's role in ribosome biogenesis through:

• Northern blot analysis of pre-rRNA intermediates in wild-type versus RPS18A-deficient plants

• Pulse-chase labeling of rRNA to track processing kinetics

• Co-immunoprecipitation studies to identify interactions with known ribosome assembly factors

• Subcellular localization studies to monitor nucleolar versus cytoplasmic distribution

How is RPS18A expression regulated in response to environmental stresses?

Ribosomal protein gene expression, including RPS18A, responds dynamically to environmental conditions. While global protein synthesis often decreases under stress to conserve energy, certain ribosomal proteins may show differential regulation. The Target of Rapamycin (TOR) signaling pathway appears to be a central regulator of this process.

Research indicates that TOR positively regulates the transcription of ribosomal proteins along with rRNA synthesis . Overexpression of TOR in Arabidopsis enhances tolerance to various abiotic stresses, including osmotic stress, salt stress, and drought conditions . Specifically, plants were tested under mannitol (100 mM), NaCl (150 mM), sorbitol (200 mM), and PEG (7%) treatments to simulate different stress conditions .

To methodically investigate RPS18A expression under stress conditions, researchers should:

• Apply precisely controlled stress treatments at defined concentrations and durations

• Extract RNA from treated and control plants at multiple time points

• Perform quantitative RT-PCR using RPS18A-specific primers and appropriate reference genes

• Validate expression changes at the protein level using Western blotting with specific antibodies

• Compare results with other RPS18 paralogs to identify paralog-specific responses

These approaches would reveal whether RPS18A is part of the stress-responsive translational machinery that helps plants adapt to challenging environmental conditions.

How does TOR kinase signaling affect RPS18A function?

The Target of Rapamycin (TOR) kinase pathway serves as a master regulator of cellular growth and metabolism in response to environmental conditions. In Arabidopsis, TOR controls the expression of ribosomal proteins, likely including RPS18A. Plants overexpressing TOR show increased growth, biomass, and yield under both normal and stress conditions .

TOR appears to influence ribosomal protein function through multiple mechanisms:

• Transcriptional regulation: Research has demonstrated upregulation of ribosomal protein large and small subunit (RPL and RPS) genes in AtTOR overexpressing transgenic lines . Although RPS18A was not specifically mentioned, it likely follows similar regulatory patterns as other ribosomal proteins.

• Post-translational modifications: TOR activates S6K1 (ribosomal protein S6 kinase 1), which phosphorylates various ribosomal proteins . Phosphoproteomic analysis in Arabidopsis T-DNA insertion mutants showed differential regulation in the phosphorylation of p70kDa ribosomal protein S6K1 . This phosphorylation cascade likely extends to other ribosomal proteins, potentially including RPS18A.

• Coordination with stress responses: TOR overexpression enhances tolerance to osmotic and salt stress treatments, suggesting that TOR-regulated ribosomal proteins contribute to stress adaptation mechanisms .

A detailed methodological approach to study TOR-RPS18A interactions would include:

• Generating transgenic lines with altered TOR activity

• Analyzing RPS18A expression levels and post-translational modifications

• Investigating polysome profiles to assess translation efficiency

• Examining ribosome composition in different genetic backgrounds and stress conditions

What are the optimal methods for expressing and purifying recombinant RPS18A?

Producing high-quality recombinant RPS18A requires careful selection of expression systems and purification strategies. Based on commercial practices, several approaches have proven successful:

Expression Systems:

• E. coli expression: Commonly used for ribosomal proteins due to high yield and simplicity. Optimal results typically require:

  • Codon-optimized sequences for bacterial expression

  • Fusion tags (His, GST, or MBP) to enhance solubility

  • Inducible promoters with fine-tuned expression conditions

  • Low-temperature induction (16-18°C) to improve folding

• Alternative expression systems: Commercial recombinant RPS18A is also produced in yeast, baculovirus, or mammalian cell systems . These may provide advantages for proper folding and post-translational modifications.

Purification Strategy:

• Initial capture: Affinity chromatography using the fusion tag (typically His-tag with Ni-NTA resin)

• Intermediate purification: Ion exchange chromatography to remove contaminants

• Polishing: Size exclusion chromatography for final purity and buffer exchange

Commercial preparations of recombinant RPS18A achieve ≥85% purity as determined by SDS-PAGE , indicating that high purity is attainable with standard methods. The expected molecular weight of RPS18A is approximately 17-18 kDa.

Quality Control:

• SDS-PAGE to confirm size and purity

• Western blotting using specific antibodies

• Mass spectrometry to verify protein identity and detect post-translational modifications

• Functional assays to confirm biological activity (e.g., RNA binding assays)

These methodological considerations ensure the production of high-quality recombinant RPS18A suitable for biochemical, structural, and functional studies.

What immunological techniques are most effective for detecting RPS18A in plant tissues?

Detecting endogenous RPS18A in plant tissues requires specific antibodies and appropriate immunological techniques. Commercially available rabbit polyclonal antibodies against Arabidopsis RPS18A enable several detection approaches:

Western Blotting:

• Extract total protein using a buffer containing detergents (e.g., RIPA) and protease inhibitors

• Separate proteins by SDS-PAGE (12-15% gels recommended for small proteins)

• Transfer to PVDF or nitrocellulose membrane

• Block with 5% non-fat milk or BSA in TBST

• Incubate with anti-RPS18A primary antibody (1:1000-1:5000 dilution)

• Detect with appropriate secondary antibody and visualization system

• Expected band size: approximately 17-18 kDa

Immunoprecipitation (IP):

• Prepare non-denaturing lysates from plant tissues

• Pre-clear with protein A/G beads

• Incubate with anti-RPS18A antibody

• Capture antibody-protein complexes with protein A/G beads

• Wash extensively to remove non-specific binding

• Elute and analyze by western blotting or mass spectrometry

Enzyme-Linked Immunosorbent Assay (ELISA):
The available antibodies are suitable for ELISA applications , enabling quantitative detection of RPS18A. This approach is particularly useful for:

• Screening multiple samples simultaneously

• Quantitative comparison across different conditions or genotypes

• High-throughput analysis of expression patterns

Immunohistochemistry/Immunofluorescence:
For visualizing the subcellular localization of RPS18A:

• Fix plant tissues with paraformaldehyde

• Section or permeabilize cells

• Block with appropriate serum

• Incubate with anti-RPS18A antibody

• Detect with fluorescently-labeled secondary antibody

• Counterstain nuclei and visualize by confocal microscopy

These techniques provide complementary information about RPS18A expression, localization, and interactions within plant tissues.

How conserved is RPS18A across different species?

RPS18A shows remarkable evolutionary conservation across eukaryotic kingdoms, reflecting its fundamental role in ribosome function. The ortholog information indicates that RPS18 is found in diverse organisms :

Plants:

• Arabidopsis thaliana (RPS18A, RPS18B, RPS18C)

• Oryza sativa (RPS18A, RPS18B)

• Glycine max (multiple paralogous genes)

• Populus trichocarpa

• Chlamydomonas reinhardtii

• Volvox carteri

Animals:

• Humans (RPS18)

• Mammals (mouse, rat, dog)

• Fish (Danio rerio, Oryzias latipes)

• Insects (Drosophila, Anopheles)

• Nematodes (C. elegans)

Fungi:

• Saccharomyces cerevisiae (RPS18A, RPS18B)

• Schizosaccharomyces pombe (RPS18-1, RPS18-2)

• Various other fungal species

Protists:

• Dictyostelium discoideum

• Plasmodium falciparum

• Toxoplasma gondii

This high degree of conservation across evolutionary distant organisms suggests that RPS18A performs a fundamental function in the ribosome that has been maintained throughout eukaryotic evolution. The protein's core structure and function are likely highly similar across these diverse species.

Interestingly, many organisms maintain multiple copies of RPS18 genes. Arabidopsis has three paralogs (RPS18A, RPS18B, RPS18C) , while yeasts like S. cerevisiae and S. pombe have two copies each . This pattern suggests that duplication and potential functional divergence of RPS18 genes may provide evolutionary advantages.

What functional differences exist between RPS18A and its paralogs in Arabidopsis?

Arabidopsis thaliana contains three RPS18 paralogs: RPS18A, RPS18B, and RPS18C . While maintaining high sequence similarity, these paralogs likely possess distinct functional characteristics:

Expression patterns:
The three paralogs likely show differential expression across:

• Tissues and cell types

• Developmental stages

• Stress conditions and environmental responses

Genetic interactions:
Similar to other ribosomal protein families like RPS24A and RPS24B that exhibit combined haploinsufficiency , the RPS18 paralogs might have partially overlapping functions but also unique contributions to ribosome function.

Methodological approaches to investigate paralog-specific functions:

• Comparative expression analysis:

  • RNA-seq or microarray data analysis across tissues and conditions

  • Promoter-reporter constructs to visualize tissue-specific expression

  • Paralog-specific qRT-PCR for quantitative comparison

• Genetic studies:

  • Characterization of single, double, and triple mutants

  • Complementation tests using each paralog

  • Analysis of genetic interactions with other pathways

• Biochemical analysis:

  • Paralog-specific antibodies to study protein levels and localization

  • Ribosome incorporation efficiency of different paralogs

  • Identification of paralog-specific protein interactions

• Ribosome function:

  • Ribosome profiling to identify mRNAs preferentially translated by ribosomes containing specific RPS18 paralogs

  • Translation fidelity assays to detect paralog-specific effects on accuracy

How does RPS18A contribute to plant stress tolerance mechanisms?

The potential contribution of RPS18A to stress tolerance can be inferred from research on TOR signaling and ribosomal proteins. Plants overexpressing TOR exhibit enhanced tolerance to various abiotic stresses, including osmotic stress (mannitol 100 mM, sorbitol 200 mM), salt stress (NaCl 150 mM), and drought conditions (PEG 7%) . Since TOR positively regulates ribosomal protein expression, this suggests that ribosomal proteins like RPS18A may play important roles in stress adaptation.

Several potential mechanisms explain RPS18A's contribution to stress tolerance:

Selective translation regulation:
During stress conditions, global protein synthesis typically decreases to conserve energy, but certain stress-responsive mRNAs must still be translated. RPS18A may contribute to this selective translation through:

• Direct binding to specific mRNA features

• Participation in specialized ribosomes that preferentially translate stress-relevant transcripts

• Interaction with stress-specific translation factors

Ribosome biogenesis adaptation:
Similar to RPS24's function in 18S rRNA maturation , RPS18A may help coordinate stress-induced changes in ribosome biogenesis. This could involve:

• Modulation of pre-rRNA processing efficiency

• Altered ribosome assembly pathways under stress

• Quality control of ribosomes during stress conditions

Integration with TOR signaling:
The established connection between TOR signaling, ribosomal proteins, and stress responses suggests that RPS18A may function downstream of TOR in stress adaptation pathways, potentially through:

• Phosphorylation-dependent regulation

• Altered subcellular localization under stress

• Interaction with stress-responsive signaling components

A systematic approach to investigate RPS18A's role in stress tolerance would include:

• Generating RPS18A knockout, knockdown, or overexpression lines

• Phenotypic analysis under various stress conditions

• Transcriptome and translatome profiling during stress

• Identification of stress-specific RPS18A-interacting proteins

What methodological approaches can reveal the role of RPS18A in abiotic stress responses?

To thoroughly investigate RPS18A's function in abiotic stress responses, several complementary methodological approaches should be employed:

Genetic approaches:

• Mutant analysis:

  • T-DNA insertion lines or CRISPR/Cas9-generated knockouts

  • RNAi-mediated knockdown lines

  • Overexpression lines under constitutive or inducible promoters

  • Paralog-specific manipulations to address redundancy

• Stress tolerance phenotyping:

  • Survival rates under different stress intensities

  • Growth parameters (root length, biomass, leaf area)

  • Physiological measurements (photosynthetic efficiency, water content)

  • Stress-induced developmental alterations

Molecular approaches:

• Expression analysis:

  • qRT-PCR of RPS18A and stress marker genes

  • Western blotting to monitor protein levels

  • Promoter-reporter fusions to visualize stress-responsive expression patterns

• Translational control:

  • Polysome profiling to assess global translation status

  • Ribosome profiling to identify differentially translated mRNAs

  • mRNA association with RPS18A-containing ribosomes during stress

Biochemical approaches:

• Post-translational modifications:

  • Phosphorylation status under stress conditions

  • Other modifications (ubiquitination, SUMOylation)

  • TOR-dependent modification patterns

• Protein interactions:

  • Stress-specific interactome analysis

  • Co-immunoprecipitation with stress signaling components

  • Yeast two-hybrid screening using stress-induced cDNA libraries

Cell biological approaches:

• Subcellular localization:

  • Fluorescent protein fusions to track RPS18A during stress

  • Co-localization with stress granules or processing bodies

  • Nucleolar versus cytoplasmic distribution during stress

These methodological approaches would provide comprehensive insights into how RPS18A contributes to plant stress tolerance mechanisms at multiple levels, from gene expression to protein function.

How can RPS18A be used as a tool for studying translational control?

RPS18A offers several advantages as a tool for investigating translational control mechanisms in plants:

Ribosome tagging and purification:

• Translating Ribosome Affinity Purification (TRAP):

  • Generate transgenic plants expressing epitope-tagged RPS18A (FLAG, HA, or GFP tags)

  • Isolate ribosomes via the tag under various conditions

  • Identify actively translated mRNAs by sequencing associated transcripts

  • This approach allows cell-type-specific or condition-specific translatome analysis

• Selective Ribosome Profiling:

  • Create RPS18A fusion with proximity labeling enzymes (BioID, TurboID)

  • Biotinylate proteins and RNAs near the ribosome in vivo

  • Isolate and identify these molecules to map the translational environment

Paralog-specific functions:

• Paralog replacement studies:

  • Complement rps18a mutants with each paralog or with chimeric constructs

  • Analyze resulting translatome changes

  • Identify regions responsible for paralog-specific functions

• Selective ribosome manipulation:

  • Conditionally deplete RPS18A using degron technologies

  • Monitor immediate effects on translation of specific mRNAs

  • Identify RPS18A-dependent translation events

Stress-responsive translation:

• Condition-specific translatome analysis:

  • Compare RPS18A-associated mRNAs under normal versus stress conditions

  • Identify stress-responsive mRNAs dependent on RPS18A

  • Correlate with TOR signaling activity

• Structure-function analysis:

  • Generate a panel of RPS18A variants with specific mutations

  • Analyze effects on global and specific mRNA translation

  • Identify functional domains important for stress-responsive translation

Technical considerations:

• Ensure tag fusions don't disrupt RPS18A function by complementation testing

• Include appropriate controls for ribosome purification experiments

• Use spike-in controls for quantitative comparisons between conditions

• Consider potential compensation by other paralogs (RPS18B, RPS18C)

These applications make RPS18A a valuable tool for unraveling the complex mechanisms of translational control in plants, particularly in response to developmental signals and environmental stresses.

What methods can detect and characterize RPS18A post-translational modifications?

Post-translational modifications (PTMs) of ribosomal proteins are emerging as important regulatory mechanisms affecting ribosome function. Investigating RPS18A modifications requires sophisticated methodological approaches:

Identification of PTMs:

• Mass spectrometry-based proteomics:

  • Purify RPS18A using immunoprecipitation or tagged protein isolation

  • Digest with proteases (trypsin, chymotrypsin, or multiple proteases)

  • Analyze using high-resolution LC-MS/MS

  • Search for modifications including phosphorylation, acetylation, methylation, ubiquitination, and SUMOylation

  • Use neutral loss scanning for phosphopeptide identification

• Targeted phosphoproteomic analysis:

  • Enrich phosphopeptides using TiO2, IMAC, or phospho-specific antibodies

  • Apply parallel reaction monitoring (PRM) for targeted analysis

  • Compare phosphorylation patterns across conditions and genetic backgrounds

  • Research indicates phosphorylation may be regulated by TOR signaling through S6K1

• Modification-specific antibodies:

  • Generate antibodies against predicted modification sites

  • Use for Western blotting to monitor modification status

  • Apply for immunoprecipitation of modified forms

Functional analysis of PTMs:

• Site-directed mutagenesis:

  • Create non-modifiable variants (S→A for phosphorylation, K→R for acetylation/ubiquitination)

  • Generate phosphomimetic variants (S→D/E)

  • Express in rps18a mutant background and analyze phenotypes

  • Assess effects on stress tolerance, development, and translation

• In vitro functional assays:

  • Express and purify RPS18A with defined modifications

  • Perform RNA binding assays to assess impact on RNA interactions

  • Conduct in vitro translation assays with modified and unmodified forms

  • Analyze effects on translation initiation, elongation, and termination

• Identification of modifying enzymes:

  • Screen kinases, acetyltransferases, and other modifying enzymes

  • Verify interactions and modification activity in vitro

  • Analyze genetic interactions between RPS18A and modifying enzymes

  • Investigate TOR pathway components that might regulate these enzymes

Analysis of modification dynamics:

• Temporal analysis:

  • Monitor RPS18A modifications across developmental stages

  • Track changes during stress responses

  • Correlate with alterations in translation patterns

• Spatial analysis:

  • Determine tissue-specific modification patterns

  • Analyze subcellular distribution of modified forms

  • Investigate if modifications affect nucleolar localization or cytoplasmic functions

These approaches provide a comprehensive framework for investigating how post-translational modifications regulate RPS18A function in translation and stress responses, potentially revealing novel mechanisms of translational control in plants.