Recombinant Danio rerio RRP15-like protein (rrp15)

What is Recombinant Danio rerio RRP15 and what are its basic characteristics?

Recombinant Danio rerio RRP15 is a 249-amino acid protein involved in ribosomal RNA processing. It is known by alternative names including rrp15, zgc:56269, and RRP15-like protein. The protein is primarily localized in the cytoplasm and has been identified as a component critical for nucleolar formation and ribosome biogenesis . The amino acid sequence of Danio rerio RRP15 is:
MAALAVKPHVVVEDGNDDVSEVSISNDEESGADSDHEGADAENSGDEDGKSDEEQNEENPNAGWAEAMAKVLGKKTPDTKPSILLKNKQLDKIKEKEKKERLEKKKQLDKKRAWENICREKPDVVQDREHERNLQRVATRGVVQLFNALKNHQKNVNERIKEVGGSERKKSKILSSVSKKDFIDVLRGTDVAVKKEKEIKAEKSSWSVLKDDFMMGASMKDWDKERDEDGGGGEEDEREPAQEYSSESD .

How does Danio rerio RRP15 function differ from its yeast homolog?

Unlike the budding yeast Rrp15p, which functions exclusively as a component of pre-60S ribosomal subunits, Danio rerio RRP15 (like human RRP15) is found in both pre-40S and pre-60S subunits. This suggests a broader role in ribosome biogenesis across vertebrates compared to yeast . Methodologically, researchers investigating these functional differences typically employ sucrose gradient centrifugation to separate ribosomal subunits, followed by western blotting or mass spectrometry to identify associated proteins, revealing this distinct evolutionary divergence in function.

What expression systems are recommended for producing recombinant Danio rerio RRP15?

Recombinant Danio rerio RRP15 can be successfully expressed in multiple host systems including yeast, E. coli, baculovirus, and mammalian cell expression systems . The choice of expression system depends on your experimental requirements:

For most basic research applications, E. coli-expressed protein yields sufficient purity (>85% as determined by SDS-PAGE) and functionality, though researchers should consider mammalian expression systems when studying interactions requiring native post-translational modifications .

How can Danio rerio RRP15 be utilized in cancer research models?

Danio rerio RRP15 has emerged as a significant protein in cancer research due to its involvement in nucleolar stress pathways. Studies have demonstrated that RRP15 is frequently upregulated in colorectal cancer (CRC) and hepatocellular carcinoma (HCC), with expression levels correlating with TNM stage and poor survival outcomes .

When designing zebrafish-based cancer models, researchers should consider:

• Creating transgenic zebrafish with conditional RRP15 expression to study its oncogenic potential

• Employing CRISPR/Cas9-mediated knockdown to assess effects on cancer progression in vivo

• Using xenograft models with human cancer cells modified to express zebrafish RRP15 to study cross-species conservation of oncogenic pathways

Experimental data indicates that silencing RRP15 induces ribosome stress, cell cycle arrest, and apoptosis, which collectively suppress cell proliferation and metastasis in cancer models . This makes it a promising target for developing novel cancer therapeutics.

What are the implications of RRP15's interaction with LMNA (lamin A) in Danio rerio?

The interaction between RRP15 and LMNA (lamin A) in Danio rerio, identified through affinity capture-mass spectrometry, suggests a potential link between nuclear envelope integrity and ribosome biogenesis . This interaction may provide insight into how nucleolar function is coordinated with nuclear structure.

To investigate this interaction, researchers typically employ:

• Proximity-dependent biotinylation techniques (BioID) in zebrafish embryos, which have been adapted for in vivo vertebrate studies

• Co-immunoprecipitation assays followed by western blotting

• Fluorescence microscopy with co-localization analysis

The LMNA-RRP15 interaction identified through high-throughput screening had a BFDR (Bayesian False Discovery Rate) cutoff of ≤1%, indicating high confidence in this protein-protein association . This interaction opens new avenues for understanding laminopathies and their connection to ribosome biogenesis defects.

How does RRP15 deficiency affect the Wnt/β-catenin pathway in vertebrate models?

RRP15 deficiency induces ribosome stress that suppresses the Wnt/β-catenin signaling pathway through a fascinating molecular mechanism. Research has revealed that ribosome stress caused by RRP15 knockdown facilitates the translation of TOP mRNA LZTS2 (Leucine zipper tumor suppressor 2), which leads to nuclear export and degradation of β-catenin .

The experimental approach to studying this mechanism involves:

• RRP15 silencing using siRNA or CRISPR/Cas9 in zebrafish embryos or cell lines

• Assessment of ribosome biogenesis using polysome profiling and northern blotting

• Analysis of Wnt/β-catenin pathway components through western blotting, immunofluorescence, and reporter assays

• Translational efficiency analysis of TOP mRNAs using polysome fractionation and qRT-PCR

These findings suggest RRP15 could be a therapeutic target in cancers with hyperactive Wnt signaling, particularly in high-ribosome biogenesis colorectal cancers .

What are the optimal storage and handling conditions for recombinant Danio rerio RRP15?

Recombinant Danio rerio RRP15 is typically supplied as a lyophilized powder and requires specific handling conditions to maintain structural integrity and biological activity:

After reconstitution, aliquot the protein to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce protein activity. The presence of 6% trehalose in the formulation helps maintain protein stability during freeze-thaw processes .

What methods are most effective for studying RRP15's role in nucleolar formation?

To investigate RRP15's critical role in nucleolar formation, researchers should employ a multi-faceted experimental approach:

• Immunofluorescence microscopy: Use antibodies against nucleolar markers (fibrillarin, nucleolin) alongside RRP15 to visualize co-localization and structural changes following RRP15 depletion.

• Electron microscopy: For ultrastructural analysis of nucleolar morphology changes.

• RRP15 knockdown strategies:

  • Morpholino oligonucleotides in developing zebrafish embryos

  • siRNA or shRNA in zebrafish cell lines

  • CRISPR/Cas9-mediated gene editing for stable knockout models

• Nucleolar isolation: Differential centrifugation to isolate intact nucleoli, followed by proteomic analysis to identify RRP15-dependent nucleolar composition changes.

Research has demonstrated that RRP15 is essential for nucleolar formation, and its perturbation induces nucleolar stress that activates checkpoint responses, making these methodologies crucial for understanding its fundamental role in cellular homeostasis .

How can researchers effectively measure RRP15-induced nucleolar stress in zebrafish models?

Nucleolar stress induced by RRP15 deficiency can be effectively measured using several complementary approaches in zebrafish models:

• rRNA processing analysis:

  • Northern blotting with probes targeting pre-rRNA intermediates

  • Pulse-chase labeling with 5-ethynyl uridine (EU) to track newly synthesized rRNA

• Checkpoint activation markers:

  • Western blotting for p53 stabilization and phosphorylation

  • qRT-PCR analysis of p53 target genes (p21, Bax, PUMA)

  • Immunostaining for RPL5/RPL11 localization

• Cell cycle analysis:

  • Flow cytometry with propidium iodide staining

  • EdU incorporation assays to measure S-phase entry

  • Phospho-histone H3 immunostaining to detect mitotic cells

• In vivo assessment:

  • Transgenic zebrafish lines with fluorescent cell cycle reporters

  • Whole-mount immunostaining for checkpoint proteins

  • Live imaging of nucleolar dynamics using fluorescently tagged nucleolar proteins

Research has shown that RRP15 deficiency activates the RPL5/RPL11/5S rRNA-Mdm2-p53 axis in p53-proficient cells, causing G1-G1/S arrest, while p53-deficient cells show S-phase perturbation and ATR-Chk1-γH2AX axis activation .

How can researchers address low protein yield when expressing recombinant Danio rerio RRP15?

When encountering low yield of recombinant Danio rerio RRP15, consider the following optimization strategies:

• Expression system adjustment:

  • For E. coli expression: Test multiple strains (BL21, Rosetta, Arctic Express) and optimize induction conditions (temperature, IPTG concentration, induction time)

  • For yeast expression: Evaluate different promoters and secretion signals

  • For baculovirus: Optimize MOI and harvest time

• Solubility enhancement:

  • Express as fusion protein with solubility tags (MBP, SUMO, GST)

  • Include appropriate chaperones to assist folding

  • Optimize lysis buffer composition with stabilizing agents

• Purification optimization:

  • Test multiple affinity tags for optimal purification efficiency

  • Implement two-step purification strategy for higher purity

  • Adjust buffer conditions to maintain protein stability

• Protein degradation prevention:

  • Include protease inhibitors throughout purification

  • Maintain cold chain during all processing steps

  • Determine optimal pH and salt concentration for stability

Based on published protocols, E. coli-based expression systems can achieve >85% purity as determined by SDS-PAGE when optimized correctly .

What strategies can resolve contradictory results between in vitro and in vivo RRP15 studies?

When facing discrepancies between in vitro and in vivo RRP15 studies, implement these systematic approaches:

• Validate protein functionality:

  • Confirm proper folding using circular dichroism or thermal shift assays

  • Verify activity with functional assays (e.g., RNA binding assays)

  • Check for proper post-translational modifications

• Reconcile experimental conditions:

  • Match buffer compositions between in vitro and cellular conditions

  • Consider physiological protein concentrations

  • Account for cellular compartmentalization effects

• Employ complementary approaches:

  • Perform rescue experiments with wild-type protein

  • Use domain mapping to identify functional regions

  • Conduct structure-function relationship studies

• Consider context-dependent effects:

  • Evaluate cell/tissue-specific co-factors

  • Assess developmental stage-specific functions

  • Examine microenvironment influences on protein activity

Research has shown that RRP15 functions differently in p53-proficient versus p53-deficient cellular contexts, which may explain some experimental discrepancies . Additionally, since RRP15 functions differ between yeast and vertebrates, evolutionary considerations should be incorporated when interpreting cross-species data .

How should researchers interpret differential effects of RRP15 knockdown in p53-proficient versus p53-deficient systems?

The differential effects of RRP15 knockdown between p53-proficient and p53-deficient systems represent a crucial consideration for experimental design and data interpretation:

To properly interpret these differential effects:

• Always determine p53 status in your experimental system before studying RRP15 function

• Include appropriate controls from both p53-proficient and p53-deficient backgrounds

• Monitor multiple checkpoint pathways simultaneously (p53 and ATR-Chk1)

• Validate findings across different cell types to ensure robustness

This differential response to RRP15 deficiency suggests potential selective targeting of p53-deficient cancer cells, as they undergo cell death rather than cell cycle arrest when RRP15 is depleted .

How might RRP15 be exploited as a therapeutic target in cancer based on zebrafish research models?

Based on findings from zebrafish and other vertebrate models, RRP15 shows significant potential as a therapeutic target in cancer treatment through several mechanisms:

• Selective cancer cell targeting:

  • RRP15 inhibition causes cell death specifically in p53-deficient cancer cells while inducing reversible cell cycle arrest in normal cells

  • This differential response creates a therapeutic window for cancer-specific interventions

• Metastasis inhibition pathways:

  • RRP15 knockdown reduces epithelial-to-mesenchymal transition (EMT) and inhibits migration in hepatocellular carcinoma models

  • This occurs through downregulation of the LAMC2/ITGB4/FAK pathway

• Wnt/β-catenin pathway modulation:

  • Ribosome stress induced by RRP15 deficiency suppresses Wnt/β-catenin signaling

  • This mechanism is particularly relevant for colorectal cancers which often feature hyperactive Wnt signaling

Future research should focus on developing zebrafish xenograft models with human cancer cells to test RRP15-targeting compounds, evaluating off-target effects of RRP15 inhibition on normal tissues, and developing small molecule inhibitors specific to RRP15.

What can evolutionary studies of RRP15 across vertebrates tell us about ribosome biogenesis regulation?

Evolutionary studies of RRP15 across vertebrate species, including Danio rerio, can provide significant insights into ribosome biogenesis regulation:

• Functional divergence analysis:

  • Compare RRP15 function between yeast (pre-60S specific) and vertebrates (pre-40S and pre-60S)

  • Identify conserved domains versus species-specific regions to determine core functionality

• Interaction network evolution:

  • Map RRP15 protein-protein interactions across species using affinity purification-mass spectrometry

  • Determine which interactions are evolutionarily conserved versus species-specific

• Regulatory mechanism comparison:

  • Analyze promoter regions and transcriptional control of RRP15 across species

  • Identify conserved regulatory elements that control expression timing

• Disease-relevant variations:

  • Correlate species-specific variations with differential disease susceptibility

  • Identify potential compensatory mechanisms in different vertebrate models

Understanding the evolutionary trajectory of RRP15 function can provide insights into the fundamentals of ribosome biogenesis regulation and reveal potential species-specific vulnerabilities relevant to disease and therapeutic development.

How can advanced imaging techniques advance our understanding of RRP15 dynamics in zebrafish development?

Advanced imaging techniques offer powerful approaches to elucidate RRP15 dynamics during zebrafish development:

• Super-resolution microscopy applications:

  • Stimulated emission depletion (STED) microscopy to visualize nucleolar subcompartments

  • Photoactivated localization microscopy (PALM) to track single molecules of RRP15 in live cells

  • Structured illumination microscopy (SIM) for dynamic nucleolar reorganization

• Live imaging in zebrafish embryos:

  • Generate transgenic zebrafish with fluorescently tagged RRP15

  • Perform light sheet microscopy for long-term, low-phototoxicity imaging

  • Use selective plane illumination microscopy (SPIM) for whole-embryo visualization

• Multi-modal imaging approaches:

  • Combine fluorescence imaging with electron microscopy using correlative light and electron microscopy (CLEM)

  • Implement expansion microscopy for high-resolution imaging of nucleolar components

  • Utilize lattice light sheet microscopy for high-speed 3D imaging

• Functional imaging techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure RRP15 mobility

  • Förster resonance energy transfer (FRET) to detect protein-protein interactions

  • Fluorescence lifetime imaging microscopy (FLIM) to map RRP15 interaction dynamics