RPL23A Human

What is RPL23A and what is its basic function in human cells?

RPL23A (Ribosomal Protein L23a) is a component of the 60S subunit in cytoplasmic ribosomes, which are responsible for protein synthesis. It belongs to the universal ribosomal protein uL23 family and is primarily located in the cytoplasm . As part of the large ribosomal subunit, RPL23A contributes to the structure and function of the ribosome, which consists of 4 rRNA species and approximately 80 structurally distinct proteins that collectively catalyze protein synthesis .

Beyond its canonical role in ribosomes, RPL23A may be involved in mediating growth inhibition by interferon and has been implicated in promoting p53/TP53 degradation through stimulation of MDM2-mediated TP53 polyubiquitination, suggesting roles in cellular growth regulation and cell cycle control .

What is the genomic organization of RPL23A and its related pseudogenes?

The RPL23A gene is co-transcribed with several small nucleolar RNA genes - U42A, U42B, U101A, and U101B - which are located in its third, first, second, and fourth introns, respectively . This co-transcriptional arrangement may represent a regulatory mechanism ensuring coordinated expression of these genes that potentially function together in ribosome biogenesis.

A remarkable feature of RPL23A is its extensive pseudogene family, with 95 related pseudogenes identified (RPL23AP1 through RPL23AP97, excluding RPL23AP9 and RPL23AP13) . This large number of pseudogenes is typical for genes encoding ribosomal proteins and provides insights into the evolutionary history of the human genome. Some pseudogenes, such as RPL23AP53, have been shown to be transcriptionally active and may have biological significance .

How can RPL23A be studied experimentally in laboratory settings?

Several experimental approaches have been employed to study RPL23A:

Gene Expression Analysis:

• Quantitative RT-PCR using specific primers for RPL23A or its pseudogenes (e.g., RPL23AP53)

• RNA-seq for genome-wide expression analysis and correlation studies

Protein Analysis:

• Western blotting using specific antibodies (e.g., ab157110) for detecting RPL23A protein

• Immunoprecipitation for studying protein-protein interactions

Functional Studies:

• cDNA cloning and recombinant protein expression in E. coli using expression vectors like pET28a

• Protein purification via Ni chelating affinity chromatography for His-tagged RPL23A

• Cell proliferation assays (e.g., MTT assay) to assess effects on cell growth

Pathway Analysis:

• Gene Set Enrichment Analysis (GSEA) for identifying pathways associated with RPL23A expression

• REACTOME pathway analysis for understanding cellular processes linked to RPL23A

These approaches can be combined to comprehensively investigate the structure, expression, interactions, and functions of RPL23A in various cellular contexts.

What is the relationship between RPL23A and disease states?

RPL23A has been implicated in several disease processes:

Autoimmune Disorders:

RPL23A has been identified as an autoimmune target causing a form of rheumatoid arthritis in mice and eliciting reactions from T cells and autoantibodies from human rheumatoid arthritis patients . This suggests RPL23A may be recognized as a self-antigen in certain autoimmune conditions.

Cancer:

Recombinant RPL23A protein exhibits anti-cancer activity against Hep-2 cells (laryngeal cancer cells) with a time- and dose-dependent effect . Interestingly, lower concentrations (0.185 μg/mL) showed better growth inhibition rates (up to 36.31%) than higher concentrations, suggesting a potential tumor suppressor role .

Melanoma:

The RPL23A pseudogene, RPL23AP53, shows differential expression between primary and metastatic melanoma samples, with significant differences also observed according to nodal status (N0 vs. N3, N1 vs. N3) . This suggests a potential role in cancer progression or utility as a biomarker.

How does the structure of RPL23A relate to its function?

The RPL23A protein has a molecular weight of approximately 17.719 kDa with a theoretical pI of 11.16 . In recombinant systems, fusion with His-tags increases the molecular weight to approximately 21-22 kDa .

While the search results don't provide detailed structural information, we know that RPL23A binds to specific sites on ribosomal RNA, as evidenced by its yeast counterpart binding to a specific site on the 26S rRNA . This RNA-binding capability is essential for ribosomal assembly and stability.

The high conservation of RPL23A across species suggests structural elements critical for its function in ribosomes. For example, in Arabidopsis, paralogous genes RPL23aA and RPL23aB encode proteins with 95% amino acid identity, and their proteins are functionally equivalent despite differential expression levels .

What is known about the regulation of RPL23A expression?

Direct information about human RPL23A expression regulation is limited in the search results, but we can draw insights from studies of related genes:

In plants (Arabidopsis), paralogous RPL23a genes are expressed in a concerted manner, with one paralog (RPL23aA) expressed at much higher levels than the other (RPL23aB) across different developmental stages and tissues . This suggests coordinated regulation mechanisms.

The co-transcription of RPL23A with several small nucleolar RNA genes located in its introns (U42A, U42B, U101A, and U101B) suggests an intricate regulatory mechanism that ensures proper stoichiometry of ribosomal components .

Studies in melanoma cells have shown that RPL23AP53 (a pseudogene) expression varies between primary and metastatic samples and correlates with certain clinical parameters , indicating that even pseudogenes derived from RPL23A may be subject to context-dependent regulation.

How do RPL23A pseudogenes differ functionally from the canonical gene?

While pseudogenes typically lack functional elements required for proper protein expression, some RPL23A pseudogenes appear to be transcriptionally active and may have acquired novel functions:

RPL23AP53 has been extensively studied in melanoma, where its expression significantly differs between primary and metastatic samples . Analysis using tools like REACTOME pathway browser and Gene Set Enrichment Analysis (GSEA) reveals that RPL23AP53 expression correlates with specific cellular phenotypes and pathways .

The expression of RPL23AP53 has been correlated with BRAF status in melanoma patients, particularly in BRAF wild-type groups, suggesting potential regulatory interactions or parallel regulation .

These findings suggest that some RPL23A pseudogenes may function as regulatory RNAs influencing gene expression or as competitors for factors that interact with the functional RPL23A gene, representing an evolution from mere genomic "fossils" to functional genetic elements.

What experimental challenges arise when studying RPL23A?

Several experimental challenges can be anticipated when studying RPL23A:

Pseudogene Interference:

With 95 related pseudogenes, designing specific primers and probes requires careful consideration to avoid cross-reactivity . For example, when studying RPL23AP53, researchers used carefully designed primers (5'-GAA GAT CCG CAT GTC ACT CA-3′ and 5′-TGG TCA GCG GAA ACT TGA TA-3′) and verified them using NCBI BLAST .

Functional Redundancy:

In systems with paralogous genes (as demonstrated in Arabidopsis), functional redundancy can mask phenotypes in single gene knockout/knockdown experiments . Research showing that RPL23aA and RPL23aB proteins are functionally equivalent yet expressed at different levels illustrates the complexity of studying gene function in redundant systems .

Extra-Ribosomal Functions:

Distinguishing between canonical ribosomal functions and extra-ribosomal functions (such as p53 regulation) requires specific experimental designs that can separate these roles .

Protein Purification:

Purifying active recombinant RPL23A requires optimization of expression systems and purification protocols. Research shows that N-terminally His-tagged RPL23A can be successfully expressed in E. coli and purified using Ni chelating affinity chromatography .

What is the relationship between RPL23A and the p53 pathway?

RPL23A has been reported to promote p53/TP53 degradation through stimulation of MDM2-mediated TP53 polyubiquitination . This suggests that RPL23A may play a regulatory role in the p53 pathway, which is central to cellular responses to stress, DNA damage, and oncogenic signals.

This interaction represents a critical extra-ribosomal function of RPL23A and provides a potential mechanism by which alterations in ribosomal proteins could contribute to cancer development. By modulating p53 stability, RPL23A could influence cell cycle progression, apoptosis, and DNA repair processes.

The connection between RPL23A and p53 also suggests a link between ribosome biogenesis, translation control, and cell cycle regulation, illustrating the intricate network of cellular pathways that coordinate growth and division with protein synthesis capacity.

How can RPL23A be targeted therapeutically based on current research?

Current research suggests several potential therapeutic approaches involving RPL23A:

Anti-Cancer Applications:

Recombinant RPL23A protein has demonstrated anti-cancer effects on Hep-2 cells, with growth inhibition rates up to 36.31% at optimal concentrations . This suggests potential applications as a therapeutic protein or a template for developing mimetic drugs.

Autoimmune Disease Intervention:

Given its identification as an autoimmune target in rheumatoid arthritis , RPL23A could be a target for developing tolerizing therapies or diagnostics for autoimmune conditions.

Pseudogene-Based Diagnostics:

The differential expression of RPL23AP53 in melanoma subtypes suggests potential utility as a diagnostic or prognostic biomarker . Expression analysis of RPL23A pseudogenes might aid in cancer classification and treatment selection.

p53 Pathway Modulation:

The role of RPL23A in p53 degradation suggests that modulating RPL23A-MDM2-p53 interactions could provide a novel approach to activating p53 in cancer cells where this tumor suppressor is inactivated but not mutated.

What methodological approaches are most effective for studying RPL23A pseudogenes?

Based on published research, effective approaches for studying RPL23A pseudogenes include:

Expression Profiling:

Real-time quantitative RT-PCR with highly specific primers is essential for distinguishing between pseudogenes. Researchers studying RPL23AP53 used primers designed with The Universal ProbeLibrary Assay Design Center and verified by NCBI BLAST .

Correlation Analysis:

Analyzing correlations between pseudogene expression and clinical parameters or other gene expressions (e.g., correlation between RPL23AP53 and BRAF expression) can reveal functional associations .

Bioinformatic Analysis:

Utilizing tools such as REACTOME pathway browser, Gene Set Enrichment Analysis (GSEA), and Immune and ESTIMATE Scores helps identify biological processes associated with pseudogene expression .

Patient Stratification:

Grouping patients based on pseudogene expression levels (e.g., high vs. low expression quartiles) followed by survival analysis can identify potential prognostic value .

Functional Genomics:

Manipulating pseudogene expression through overexpression or knockdown approaches, followed by phenotypic assays, can directly test functional hypotheses.

What are the latest advancements in understanding RPL23A's role in cancer biology?

Recent research has revealed several important aspects of RPL23A's involvement in cancer:

Anti-Cancer Activity:

Recombinant RPL23A protein exhibits dose- and time-dependent growth inhibition effects on cancer cell lines, with optimal inhibition of Hep-2 cells at relatively low concentrations (0.185 μg/mL) . This suggests a potential tumor suppressor function that may be context-dependent.

Pseudogene Involvement:

RPL23AP53 shows differential expression between primary and metastatic melanoma samples and correlations with nodal status . This indicates that not only the canonical gene but also its pseudogenes may play roles in cancer progression.

p53 Pathway Interaction:

RPL23A's ability to promote p53/TP53 degradation through MDM2-mediated polyubiquitination provides a mechanistic link to cancer biology, as p53 is a critical tumor suppressor that regulates cell cycle arrest, apoptosis, and DNA repair.

Potential Diagnostic Applications:

Expression patterns of RPL23A and its pseudogenes may serve as biomarkers for cancer classification, progression assessment, or treatment response prediction .