Recombinant 60S ribosomal protein L15 (rpl15)

What is RPL15 and what are its primary functions in normal cells?

RPL15 (ribosomal protein L15) is a component of the 60S ribosomal large subunit that participates in the assembly process of ribosomal subunits and is involved in the processing of rRNA . In normal cells, RPL15 serves two primary functions:

• Ribosomal assembly: RPL15 is essential for the formation of the pre-60S ribosomal subunit, as demonstrated by sucrose gradient analysis showing significant reduction of pre-60S ribosomal subunits following RPL15 depletion .

• rRNA processing: Previous studies have shown that RPL15 participates in rRNA processing at the ITS1 site, affecting rRNAs required for both 40S and 60S ribosomal subunit assembly .

Deletion of RPL15 leads to abnormalities in the biogenesis of ribosomal subunits, highlighting its critical role in normal cellular function .

Where is RPL15 localized within cells?

RPL15 displays a distinct localization pattern within human cells. Immunofluorescence assays reveal that RPL15 is:

• Dispersed throughout the cytoplasm and nucleoplasm

• Concentrated in the nucleolus

Co-fluorescence imaging and Pearson correlation coefficient (R value) analysis demonstrates that RPL15 is:

• Colocalized with Bip (a rough ER marker) in the cytoplasm (but not with α-tubulin)

• Colocalized with nucleolin (nucleolar granular component marker), fibrillarin (nucleolar dense fibrillar component marker), and UBF (nucleolar fibrillar center marker) in the nucleoli

Comparative analysis with other ribosomal proteins shows that RPL15 is more concentrated in the nucleolus than RPL11 or RPS6. Subcellular fractionation confirmed this observation, with a significantly higher percentage of nuclear-localized RPL15 compared to RPL11 .

How does RPL15 contribute to nucleolar structure maintenance?

RPL15 plays a critical role in maintaining nucleolar structure. Experimental depletion of RPL15 using specific siRNAs results in:

• Increased nucleolar area relative to nuclear area

• Decreased fluorescent density of nucleolin

• Expansion of the peripheral nucleolar structure (granular component region)

• Defects in the intrinsic nucleolar structure (dense fibrillar component and fibrillar center regions)

These structural changes were quantitatively characterized using specific image-processing algorithms measuring the area of observed nucleoli and nucleus based on nucleolin and DAPI signals. Similar effects were observed with fibrillarin and UBF staining, confirming that the entire nucleolar structure becomes less compact after RPL15 depletion .

How is RPL15 expression altered in cancer, and what is its prognostic significance?

RPL15 shows altered expression across various cancer types:

• Hepatocellular carcinoma (HCC):

  • Significantly upregulated in HCC tissues and cell lines compared to normal tissues and cells

  • Strong nuclei staining in HCC tissues with little positive staining in normal tissues

  • Analysis of TCGA data confirms significant increase in RPL15 transcription in HCCs (n = 369) versus normal tissues (n = 160)

• Colon cancer:

  • Remarkably upregulated in human primary colon cancer tissues and cultured cell lines

  • Elevated expression closely correlates with clinicopathological characteristics

• Other cancers with elevated RPL15:

  • Esophageal cancer

  • Gastric cancer

• Cancers with decreased RPL15:

  • Skin squamous cell carcinoma

  • Pancreatic cancer

What molecular mechanisms underlie RPL15's role in cancer progression?

RPL15 contributes to cancer progression through multiple molecular mechanisms:

• Cell cycle regulation:

  • RPL15 overexpression promotes cell cycle progression from G1 to S phase

  • RPL15 knockdown induces cell cycle arrest in G1 phase

  • RPL15 overexpression increases CDK2 and Cyclin E expressions, while RPL15 silencing decreases these cell cycle regulators

• RPs-MDM2-p53 pathway:

  • RPL15-mediated oncogenic transformation involves the RPs-MDM2-p53 pathway

  • In response to ribosome stress, other ribosomal proteins like RPL5 and RPL11 interact with Mdm2 and inhibit its E3 ligase activity, thereby increasing p53 protein stability and transcriptional activity

• Epithelial-mesenchymal transition (EMT):

  • HCC cell invasion and migration are associated with EMT processes that are regulated by RPL15

• Differential effects in cancer vs. normal cells:

  • RPL15 depletion causes apoptosis in colon cancer cells but only cell cycle arrest in non-transformed human epithelium cells

  • This differential response suggests potential for targeted therapy

How does experimental manipulation of RPL15 affect cancer cell phenotypes?

The effects of RPL15 manipulation have been extensively studied in cancer cells:

• Effects of RPL15 silencing in HCC cells:

  • Arrested cell cycle

  • Suppressed colony formation

  • Reduced proliferation

  • Inhibited invasion and migration

  • Induced cell apoptosis

  • Suppressed xenograft tumor growth in vivo

• Effects of RPL15 overexpression in HCC cells:

  • Enhanced proliferative capacity

  • Increased colony formation

  • Promoted cell cycle progression from G1 to S phase

• Differential effects in colon cancer vs. normal cells:

  • Depletion of RPL15 causes ribosomal stress

  • This stress results in apoptosis in colon cancer cells

  • The same stress causes only G1-G1/S cell cycle arrest in non-transformed human epithelium cells

These differential responses suggest that RPL15 may serve as a promising therapeutic target with potential selectivity for cancer cells.

What techniques are most effective for studying RPL15 expression in tissue samples?

Multiple complementary techniques provide comprehensive analysis of RPL15 expression:

• RNA expression analysis:

  • RT-qPCR: Using primers (5'-GATTCGTGTTCGCCGTGGT-3' and 5'-TGCTTGTGGACTGGTTTGG-3' for RPL15) with β-actin as standardization control

  • Data analysis using the 2^-ΔΔCt method

• Protein expression analysis:

  • Western blot: Using specific antibodies against RPL15

  • Immunohistochemistry: For tissue samples, allowing visualization of cellular localization (nuclei staining pattern in cancer tissues)

• Bioinformatic analysis:

  • Mining public databases like TCGA for RPL15 expression across large cohorts

  • Kaplan-Meier survival analysis to correlate expression with patient outcomes

For comprehensive assessment, researchers should employ at least two independent methods to confirm expression changes, ideally combining RNA and protein-level analyses.

How can researchers effectively manipulate RPL15 expression in experimental models?

Several approaches have been validated for manipulating RPL15 expression:

• RPL15 knockdown:

  • siRNA transfection: Using specific RPL15 siRNAs (siRPL15-1 or -2) with RNAiMAX Transfection Reagent

  • Confirmation of knockdown efficiency by western blot and/or RT-qPCR

• RPL15 overexpression:

  • Transfection of RPL15 expression vectors in target cells (e.g., Hep3B cells)

  • Verification of overexpression by western blot

• Controls:

  • Nonsense siRNA (NS siRNA) as negative control for knockdown experiments

  • Empty vector for overexpression studies

  • Verification of endogenous protein levels by western blot

For in vivo studies, established cancer cell lines with stable RPL15 knockdown or overexpression can be used for xenograft models in immunodeficient mice .

What assays are most informative for evaluating the functional consequences of RPL15 manipulation?

Multiple assays provide comprehensive assessment of RPL15's functional roles:

• Cell proliferation assays:

  • MTT assay: Cells (3000 cells/well) transfected with or without RPL15 siRNA are analyzed at indicated timepoints

  • Colony formation assay: Evaluates long-term proliferative capacity

• Cell cycle analysis:

  • Flow cytometry: Cells fixed in 70% ethanol/30% PBS, stained with propidium iodide buffer

  • Analysis of >5,000 cells per condition using a FACSort

  • BrdU incorporation: Immunostaining with mouse α-BrdU to measure DNA synthesis

• Cell migration and invasion assays:

  • Transwell assays to evaluate metastatic potential

  • Analysis of EMT markers by western blot

• Apoptosis analysis:

  • Flow cytometry with Annexin V/PI staining

  • Evaluation of apoptotic markers (cleaved caspase-3, PARP) by western blot

• Ribosome biogenesis analysis:

  • Sucrose gradient analysis by ultracentrifugation to examine assembly of pre-ribosomal subunits

  • Analysis of pre-rRNA processing by northern blot or RT-qPCR

• Nucleolar structure analysis:

  • Immunofluorescence of nucleolar markers (nucleolin, fibrillarin, UBF)

  • Quantitative image-processing algorithms to measure nucleolar area relative to nuclear area

How should researchers interpret contradictory findings on RPL15 expression across different cancer types?

Current literature shows that RPL15 expression varies across cancer types, with upregulation in some (HCC, colon, esophageal, gastric cancers) and downregulation in others (skin squamous cell carcinoma, pancreatic cancer) . When interpreting these seemingly contradictory findings, researchers should consider:

• Tissue-specific ribosome heterogeneity:

  • Different tissues may require distinct ribosomal compositions

  • Cancer-specific alterations in ribosome composition may vary by tissue origin

• Context-dependent functions:

  • RPL15 may interact with different molecular pathways depending on the cellular context

  • The RPs-MDM2-p53 pathway may function differently across tissue types

• Technical considerations:

  • Standardize analysis methods across studies

  • Ensure appropriate normalization controls for each tissue type

  • Consider both mRNA and protein expression analyses

• Experimental validation:

  • Perform functional studies in each cancer type to confirm biological relevance

  • Use multiple cell lines representing each cancer type

Researchers should examine RPL15 expression in the context of the specific cancer type they are studying, rather than generalizing findings across all cancers.

What statistical approaches are most appropriate for correlating RPL15 expression with clinical outcomes?

When analyzing the relationship between RPL15 expression and clinical outcomes, researchers should employ robust statistical methods:

How might RPL15 serve as a therapeutic target for cancer treatment?

The differential effects of RPL15 depletion on cancer versus normal cells suggest promising therapeutic potential:

• Targeted approaches:

  • Development of specific RPL15 inhibitors that preferentially affect cancer cells

  • Exploration of RNA interference-based therapeutics targeting RPL15 mRNA

  • Investigation of molecules that disrupt RPL15's interaction with the ribosomal assembly machinery

• Combination strategies:

  • Combining RPL15 targeting with conventional chemotherapies

  • Exploring synergistic effects with other targeted therapies

  • Investigating RPL15 inhibition in the context of p53 pathway modulation

• Predictive biomarkers:

  • Using RPL15 expression levels to identify patients who might benefit from specific treatments

  • Developing companion diagnostics for RPL15-targeted therapies

• Considerations for clinical translation:

  • Tissue-specific effects must be carefully evaluated

  • Potential side effects on normal ribosome biogenesis need thorough investigation

  • Delivery methods for RPL15-targeting therapeutics require optimization

The evidence that RPL15 knockdown suppresses HCC xenograft growth in vivo and induces apoptosis specifically in cancer cells provides a strong rationale for exploring its therapeutic potential .