RPL31 Human

What is RPL31 and what is its fundamental function in human cells?

RPL31 (Ribosomal Protein L31) is a component of the large ribosomal subunit (60S) and belongs to the eukaryotic ribosomal protein eL31 family . The protein functions as an integral part of the ribosome, which is a large ribonucleoprotein complex responsible for protein synthesis in cells . RPL31 specifically contributes to the structural integrity and functional capacity of the 60S ribosomal subunit .

At the molecular level, RPL31 participates in pre-rRNA processing, which is essential for the maturation of ribosomal subunits . This processing function has significant implications for proper protein synthesis throughout the cell. The protein contains 125 amino acids in its native form and is located primarily in the cytoplasm as expected for a component of the translation machinery .

How can researchers effectively express and purify recombinant RPL31 for experimental studies?

For experimental studies requiring purified RPL31, researchers typically produce it as a recombinant protein in expression systems such as Escherichia coli . The protein can be engineered with affinity tags (commonly a 6x histidine tag at the N-terminus) to facilitate purification .

A methodological approach involves:

• Cloning the human RPL31 cDNA into an appropriate expression vector

• Transforming E. coli cells with the construct

• Inducing protein expression under optimized conditions

• Lysing cells and purifying the protein using affinity chromatography

• Performing quality control assessments using SDS-PAGE (aiming for >85% purity)

The purified protein is typically stored in a buffer containing 20mM Tris-HCl (pH8.0), 0.2M NaCl, 1mM DTT, and 50% glycerol . For long-term storage, it is recommended to store the protein at -20°C with a carrier protein (0.1% HSA or BSA) and to avoid freeze-thaw cycles .

What techniques are used to monitor RPL31 expression levels in different tissues or cell types?

Researchers employ several complementary techniques to monitor RPL31 expression:

• Quantitative RT-PCR (qRT-PCR): Total RNA is reverse transcribed, and RPL31 mRNA levels are quantified using specific primers. Results are typically normalized against housekeeping genes such as GAPDH .

• Immunohistochemistry: This technique is valuable for examining RPL31 protein expression in tissue samples, including tumor and para-cancerous tissues .

• Western Blotting: Protein extracts from cells or tissues are analyzed using RPL31-specific antibodies to quantify protein levels.

• RNA-Seq: This technique provides comprehensive transcriptome analysis, allowing researchers to assess RPL31 expression in the context of the broader gene expression landscape.

When examining potential alterations in RPL31 levels across conditions, careful consideration of appropriate control samples and normalization methods is essential for reliable comparisons.

How does RPL31 influence cancer cell proliferation and migration, and what signaling pathways are involved?

RPL31 has been identified as a potential oncogenic factor in multiple cancer types. In gastric cancer research, RPL31 was found to be abundantly expressed in both tumor tissues and cell lines (AGS and MGC-803) . Experimental silencing of RPL31 in these gastric cancer cell lines significantly inhibited proliferation and migration capabilities while promoting apoptosis .

The molecular mechanism appears to involve the JAK-STAT signaling pathway. Preliminary analysis revealed that RPL31 functions as a tumor promoter by targeting this pathway . The JAK-STAT pathway is a key regulator of cellular processes including proliferation, differentiation, and apoptosis, suggesting that RPL31 may exert its oncogenic effects through modulation of these critical cellular functions.

In vivo experiments further supported these findings, as RPL31-knockdown in gastric cancer cells inhibited xenograft tumor growth in mice . This suggests that targeting abnormally high expression of RPL31 in gastric cancer may represent a potential therapeutic strategy.

What is the relationship between RPL31 and hormone-resistant prostate cancer?

Research using shRNA-mediated functional screening identified RPL31 as a gene involved in bicalutamide-mediated effects on LNCaP prostate cancer cells . Bicalutamide is an antiandrogen used in hormone therapy for prostate cancer, but many patients eventually develop resistance.

Key findings about RPL31 in prostate cancer include:

• RPL31 is essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR) cells .

• RPL31 mRNA is more abundantly expressed in BicR cells compared to parental LNCaP cells .

• Clinical data from ONCOMINE and The Cancer Genome Atlas showed that RPL31 is overexpressed in prostate carcinomas compared to benign prostate tissues .

• RPL31 knockdown leads to increased protein levels of the tumor suppressor p53 and its targets, p21 and MDM2 .

• Decreased degradation of p53 protein was observed after RPL31 knockdown .

• The suppression of growth and cell cycle upon RPL31 knockdown was partially recovered with p53 siRNA treatment .

These findings suggest that RPL31 contributes to bicalutamide resistance in prostate cancer cells potentially through regulation of the p53 pathway, making it a possible therapeutic target for advanced prostate cancer.

How is RPL31 involved in pre-rRNA processing disorders, particularly Diamond Blackfan anemia (DBA)?

RPL31 has been identified as a novel gene linked to Diamond Blackfan anemia (DBA), a syndrome characterized by anemia and various physical abnormalities . DBA belongs to a group of related inherited bone marrow failure syndromes (IBMFS) with overlapping clinical features.

The connection between RPL31 and DBA was established through the discovery of a deletion on chromosome 2 encompassing the RPL31 gene in a patient presenting with severe macrocytic anemia, neutropenia, and multiple congenital anomalies including bilateral proximal radioulnar synostosis and a triphalangeal thumb .

Mechanistically, RPL31, like other ribosomal proteins affected in DBA, is required for pre-rRNA processing . This process involves the maturation of rRNA in pre-60S ribosomal subunits. Researchers developed a novel functional assay based on capillary electrophoresis to monitor this process, which can be used as a diagnostic tool for DBA .

The effects of decreased RPL31 expression on ribosome synthesis and erythroid differentiation validate RPL31 as a DBA gene . This discovery reveals a novel feature of the 60S subunit pre-rRNA processing pathway that can be evaluated in diagnostic laboratories, potentially aiding in the diagnosis of 20-25% of patients with DBA .

What experimental approaches are most effective for manipulating RPL31 expression in research studies?

Researchers have successfully employed several techniques to manipulate RPL31 expression:

• Lentiviral transfection: Used to construct RPL31-knockdown cell models in gastric cancer research .

• Short hairpin RNA (shRNA)-mediated screening: Employed for functional screening to identify genes involved in bicalutamide-mediated effects on prostate cancer cells .

• Small interfering RNA (siRNA)-mediated knockdown: Utilized to examine the effects of RPL31 depletion on cell proliferation, cell cycle progression, and p53 pathway activity .

• CRISPR-Cas9 genome editing: While not explicitly mentioned in the provided search results, this technology represents a state-of-the-art approach for generating more precise genetic modifications of RPL31.

When designing RPL31 knockdown experiments, researchers should consider:

• Target specificity to avoid off-target effects

• Knockdown efficiency verification by qRT-PCR and western blot

• Appropriate control conditions (e.g., scrambled shRNA/siRNA)

• Cell type-specific responses to RPL31 manipulation

• Functional validation through multiple complementary assays

What methods are used to detect RPL31 mutations or deletions in clinical samples?

For genomic analysis of RPL31 alterations, researchers employ multiple complementary techniques:

• Direct sequencing by PCR: RPL31 target regions are amplified and directly sequenced from peripheral blood DNA . This approach can identify point mutations and small insertions or deletions.

• SNP array for copy number analysis: This technique is used to detect larger deletions or duplications involving the RPL31 gene . In the reported case study, this method identified a deletion on chromosome 2 encompassing RPL31 in a patient with DBA-like symptoms .

• Next-generation sequencing: While not explicitly mentioned in the search results, targeted NGS panels or whole exome/genome sequencing represent advanced approaches for comprehensive mutation detection.

• MLPA (Multiplex Ligation-dependent Probe Amplification): Another technique that can be used to detect deletions or duplications.

For clinical applications, it's important to establish confidence intervals for detected variants, which can be calculated using methods such as the modified Wald method .

How does RPL31 expression correlate with clinical features and prognosis in cancer patients?

Research has revealed significant correlations between RPL31 expression and clinical parameters in cancer patients. In gastric cancer specifically, RPL31 expression was found to be positively correlated with the extent of tumor infiltration . This finding suggests that RPL31 may serve as a potential biomarker for disease progression and invasiveness in gastric cancer.

Aberrant expression of RPL31 has been reported across several human cancers, indicating a potential common mechanism of RPL31 involvement in carcinogenesis . In prostate cancer, clinical data from ONCOMINE and The Cancer Genome Atlas demonstrated that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues .

While specific survival data was not provided in the search results, the association between RPL31 and aggressive cancer features suggests that it may have prognostic value. Further research is needed to establish whether RPL31 expression levels could serve as an independent prognostic factor in various cancer types.

What are the hematological and developmental manifestations of RPL31 deficiency?

The clinical presentation of RPL31 deficiency has been documented in a case of Diamond Blackfan anemia (DBA) caused by a deletion encompassing the RPL31 gene . The key manifestations included:

Hematological Abnormalities:

• Severe macrocytic anemia (Hemoglobin 2 g/dL, MCV 130 fL)

• Neutropenia (absolute neutrophil counts averaging 300/μL)

• Variably elevated platelet counts (range 3×10⁵–8×10⁵/μL)

• Bone marrow findings: decreased cellularity (50-60%), absence of erythroid precursors, myeloid hypoplasia with maturation arrest, increased megakaryocytes, lymphocytosis, and eosinophilia

Developmental Abnormalities:

• Bilateral proximal radioulnar synostosis (RUS)

• Left triphalangeal thumb

• Right thenar hypoplasia

• Small spleen (2.5 cm; reference mean length for age is 5.2 cm)

These findings highlight the dual role of RPL31 in both hematopoiesis and development, similar to other ribosomal proteins implicated in DBA. The presence of both hematological and skeletal abnormalities is characteristic of ribosomopathies like DBA.

How can pre-rRNA processing analysis be used as a diagnostic tool for RPL31-related disorders?

Pre-rRNA processing analysis represents a valuable diagnostic tool for RPL31-related disorders, particularly Diamond Blackfan anemia. Researchers have developed a novel functional assay based on capillary electrophoresis measurement of rRNA maturation in pre-60S ribosomal subunits .

The methodology involves:

• Isolating RNA from patient cells (typically peripheral blood)

• Analyzing pre-rRNA processing patterns using capillary electrophoresis

• Identifying specific abnormalities in the maturation of rRNA in pre-60S ribosomal subunits

• Comparing results to patterns associated with known ribosomal protein deficiencies

This approach offers several advantages over traditional diagnostic methods:

• It is readily amenable to use in diagnostic laboratories

• It can detect functional defects in ribosome biogenesis even when genetic testing is inconclusive

• It may help identify patients with DBA who have mutations in genes not previously linked to the disorder

Research has shown that depletion of RPL31 mimics the pre-rRNA processing abnormality observed in patient cells, validating the specificity of this approach . This technique has been instrumental in establishing RPL31 as a novel DBA gene and may help diagnose approximately 20-25% of patients with DBA .

What potential therapeutic strategies could target RPL31 in cancer treatment?

Based on the established role of RPL31 in promoting cancer cell proliferation and migration, several therapeutic strategies could potentially target RPL31:

• RNA interference approaches: siRNA or shRNA targeting RPL31 has shown promising results in inhibiting cancer cell growth in experimental models . The development of clinically viable RNA interference delivery systems could translate these findings to therapeutic applications.

• Targeting the JAK-STAT pathway: Since RPL31 appears to function through the JAK-STAT signaling pathway in gastric cancer , inhibitors of this pathway might be particularly effective in cancers with RPL31 overexpression.

• Combination with p53 pathway modulators: The relationship between RPL31 and p53 in prostate cancer cells suggests that combining RPL31 inhibition with therapies that activate p53 might have synergistic effects.

• Specific inhibitors of RPL31 function: Although not yet developed, small molecules that specifically disrupt RPL31's role in ribosome biogenesis or its interaction with other proteins could provide selective targeting.

In gastric cancer specifically, inhibition of abnormally high expression of RPL31 has been proposed as a potential therapeutic strategy . For hormone-resistant prostate cancer, targeting RPL31 might help overcome resistance to antiandrogens like bicalutamide .

What are the most promising techniques for studying RPL31's role in ribosome assembly and function?

Advanced techniques that hold promise for elucidating RPL31's role in ribosome assembly and function include:

• Cryo-electron microscopy (Cryo-EM): This technique can provide high-resolution structural information about RPL31's position and interactions within the ribosome.

• Ribosome profiling: This approach can reveal how RPL31 deficiency affects translation efficiency of specific mRNAs, potentially explaining tissue-specific pathologies.

• CRISPR-Cas9 genome editing: Creating precise modifications in RPL31 can help determine which domains or residues are critical for its various functions.

• Proximity labeling techniques (BioID or APEX): These methods can identify proteins that interact with RPL31 during ribosome assembly or function.

• Single-molecule fluorescence microscopy: This can track the dynamics of ribosome assembly and translation in the presence or absence of functional RPL31.

• Mass spectrometry-based proteomics: This approach can identify post-translational modifications of RPL31 and how they affect its function.

• Capillary electrophoresis analysis of pre-rRNA processing: As demonstrated in the research on Diamond Blackfan anemia, this technique can monitor specific steps in ribosome maturation affected by RPL31 deficiency .

How might research on RPL31 contribute to our broader understanding of ribosomopathies?

Research on RPL31 has significant implications for our understanding of ribosomopathies:

• Expanding the genetic landscape: The identification of RPL31 as a novel DBA gene expands the spectrum of ribosomal proteins implicated in this disorder, potentially revealing common pathogenic mechanisms.

• Tissue specificity insights: Understanding why RPL31 deficiency primarily affects erythroid progenitors and certain developmental processes could explain the puzzling tissue specificity of ribosomopathies.

• Diagnostic advances: The pre-rRNA processing analysis developed for RPL31-related DBA represents a functional diagnostic approach that could be applied to other ribosomopathies.

• Therapeutic development: Knowledge of how RPL31 deficiency disrupts ribosome biogenesis may inform the development of targeted therapies for DBA and related disorders.

• Cancer connections: The link between RPL31 and cancer highlights the dual role of ribosomal proteins in both congenital disorders and malignancy, potentially revealing common therapeutic targets.

Understanding how mutations in a ubiquitous component of the ribosome like RPL31 can lead to such specific clinical manifestations remains a central question in ribosomopathy research. Continued study of RPL31 is likely to provide valuable insights into this paradox.

What control experiments should be included when investigating RPL31 function in cellular models?

When designing experiments to investigate RPL31 function, researchers should include the following controls:

• Appropriate knockdown controls: Use of scrambled siRNA/shRNA sequences or non-targeting CRISPR guides alongside RPL31-targeting constructs.

• Rescue experiments: Re-expression of wild-type RPL31 in knockdown/knockout cells to confirm specificity of observed phenotypes.

• Multiple cell lines: Testing effects in multiple cell types to distinguish cell-type-specific from general functions.

• Dose-dependent effects: Using varying levels of knockdown to assess the relationship between RPL31 levels and phenotypic outcomes.

• Time course analysis: Examining both immediate and delayed effects of RPL31 manipulation.

• Pathway validation: When a signaling pathway like JAK-STAT or p53 is implicated, include experiments that directly manipulate these pathways to confirm their involvement.

• Off-target effect controls: Confirmation of key findings using multiple siRNA/shRNA sequences or CRISPR guides targeting different regions of RPL31.

• Functional validation: For pre-rRNA processing studies, include known ribosomal protein deficiencies as positive controls .

These control experiments are essential for establishing the specificity and relevance of RPL31's roles in various cellular processes.

What are the key methodological considerations when analyzing RPL31 genetic variants in patient samples?

When analyzing RPL31 genetic variants in patient samples, researchers should consider:

• Comprehensive variant detection: Employ multiple methods including direct sequencing for point mutations and SNP array or MLPA for copy number variants .

• Validation of variants: Confirm detected variants using orthogonal methods and determine their frequency in control populations.

• Functional assessment: Evaluate the impact of variants on pre-rRNA processing using capillary electrophoresis or other functional assays .

• Parental testing: Determine whether variants are de novo or inherited, which may inform recurrence risk and clinical significance.

• Variant interpretation guidelines: Follow ACMG/AMP guidelines for classifying variants as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign.

• Phenotype correlation: Assess whether the clinical presentation is consistent with known RPL31-associated phenotypes, particularly in DBA .

• Conservation analysis: Evaluate evolutionary conservation of affected amino acids or nucleotides to inform potential functional importance.

• In silico prediction tools: Use computational tools to predict the impact of missense variants on protein structure and function.

• RNA studies: Consider RNA analysis to detect potential splicing effects of intronic or synonymous variants.

These methodological considerations are essential for accurate interpretation of RPL31 variants and appropriate clinical management of affected individuals.