Recombinant Danio rerio WD repeat-containing protein 55 (wdr55)

What is the basic structure and function of WDR55 protein in zebrafish?

WDR55 is a 387 amino acid protein containing six tryptophan-aspartate-repeat (WDR) motifs that form a propeller-like structure characteristic of WDR proteins . The protein sequence begins with "MATPTEHEDLSEQEVTEDEFKTPKIRETPEDIKLEAIVNTIAFHPKQDIL" and contains multiple WD40 repeats that create protein-protein interaction domains .

Functionally, WDR55 is a nucleolar protein involved in ribosomal RNA (rRNA) biosynthesis and cell cycle regulation . It modulates the production of rRNA in the nucleolus and affects cell cycle progression through the G1 phase. The protein is predicted to be located in the nucleolus and is involved in several developmental processes including chordate pharynx development, swim bladder development, and thymus development .

How conserved is WDR55 across species?

WDR55 is highly conserved across vertebrates, demonstrating its evolutionary importance. The zebrafish WDR55 protein shares approximately 66% amino acid identity with medaka fish WDR55, while showing 58% and 59% identity with human and mouse WDR55 respectively . This conservation suggests critical functional roles that have been maintained throughout vertebrate evolution.

The protein belongs to the WDR55/POC1 family (IPR050505) and contains the characteristic WD40 repeat-like-containing domain superfamily (IPR015943) . BLAST searches have confirmed that there are no other WDR55-like loci in the genomes of zebrafish, medaka, mouse, or human, indicating that WDR55 is a single-copy gene across these species .

What expression patterns does wdr55 show during zebrafish development?

While comprehensive expression data for wdr55 in zebrafish is limited, records indicate that there is at least one expression pattern figure available from Iwanami et al., 2008 . Based on its characterized functions, wdr55 expression would be expected in developing tissues that require high rates of protein synthesis and cell proliferation, particularly the thymus, pharynx, and swim bladder.

The gene's essential role in development is highlighted by studies showing that WDR55 deficiency in zebrafish causes developmental defects, particularly in thymus development, similar to observations in medaka fish where WDR55 mutation in the hokecha (hkc) mutant results in failure of thymus primordium to accumulate lymphoid cells .

What are the optimal storage and handling conditions for recombinant Danio rerio WDR55 protein?

For optimal storage and handling of recombinant Danio rerio WDR55 protein:

• Storage duration depends on formulation:

  • Liquid form generally has a shelf life of 6 months at -20°C/-80°C

  • Lyophilized form typically maintains stability for 12 months at -20°C/-80°C

• Reconstitution protocol:

  • Centrifuge vial briefly before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Default recommended final glycerol concentration is 50%

• Handling precautions:

  • Avoid repeated freezing and thawing cycles

  • Store working aliquots at 4°C for up to one week

  • For experiments requiring consistent protein activity, prepare single-use aliquots

The purity of commercially available recombinant Danio rerio WDR55 is typically >85% as determined by SDS-PAGE methods .

What approaches can be used to study wdr55 function in zebrafish models?

Multiple experimental approaches can be employed to study wdr55 function in zebrafish:

• Genetic manipulation:

  • CRISPR/Cas9 gene editing to generate targeted mutations

  • Morpholino knockdown for transient suppression of wdr55 expression

  • Transgenic overexpression of wild-type or mutant wdr55

• Phenotypic analysis:

  • Examination of thymus, pharynx, and swim bladder development

  • Assessment of cell proliferation in developing tissues

  • Analysis of rRNA processing and nucleolar structure

• Cellular localization studies:

  • EGFP-tagged WDR55 expression to visualize subcellular localization

  • Co-localization studies with nucleolar markers like fibrillarin

  • Comparison of wild-type versus mutant WDR55 localization

• Rescue experiments:

  • Introduction of wild-type wdr55 into mutant backgrounds

  • Cross-species rescue using orthologous wdr55 genes

Research has demonstrated that defects in WDR55 lead to abnormal rRNA processing and cell cycle arrest, which can be monitored using established nucleolar function assays and cell cycle analysis methods .

How can researchers effectively assess rRNA processing defects associated with wdr55 dysfunction?

To effectively assess rRNA processing defects caused by wdr55 dysfunction, researchers should consider the following methodological approaches:

• Northern blot analysis:

  • Use probes specific for different rRNA processing intermediates (e.g., 5.8S rRNA)

  • Compare patterns between wild-type and wdr55-deficient samples

  • Look specifically for accumulation of incompletely processed rRNA precursors

• Pulse-chase labeling:

  • Metabolically label newly synthesized RNA with radioactive precursors

  • Chase with non-radioactive media and analyze rRNA processing kinetics

  • Identify specific steps in rRNA processing affected by wdr55 deficiency

• RNA-seq or qRT-PCR:

  • Quantify levels of rRNA intermediates and mature rRNAs

  • Analyze expression of rRNA processing factors

  • Examine p53 pathway activation (e.g., p21 upregulation) that occurs downstream of nucleolar stress

• Polysome profiling:

  • Analyze ribosome assembly and global translation

  • Look for defects in ribosome subunit formation

Studies in cell culture models have shown that siRNA knockdown of WDR55 leads to accumulation of incompletely processed rRNA precursors while still allowing production of mature 5.8S, 18S, and 28S rRNAs, suggesting a role in specific aspects of rRNA processing rather than complete inhibition of ribosome production .

How does wdr55 dysfunction affect embryonic development in vertebrate models?

WDR55 dysfunction has profound effects on vertebrate embryonic development, with severity varying by species:

• In zebrafish and medaka:

  • Defective thymus development with failure to accumulate lymphoid cells

  • Abnormal pharyngeal and swim bladder development

  • Cell proliferation defects in developing tissues

• In Arabidopsis (plant ortholog):

  • Embryo developmental defects including failure to establish bilateral symmetry

  • Abnormal cotyledon numbers and patterns

  • Reduced apical dominance

  • Serrated early rosette leaves

• In mice:

  • WDR55-null mice are lethal before implantation, indicating an essential role in early mammalian development

These developmental effects likely stem from WDR55's role in rRNA synthesis and cell cycle progression. The nucleolar dysfunction caused by WDR55 deficiency leads to p53 activation, increased p21 expression, and cell cycle arrest at G1 phase, impacting rapidly proliferating tissues during development .

What is the evidence linking WDR55 to human disease conditions?

Several lines of evidence connect WDR55 to human disease conditions:

• Genetic studies:

  • WDR55 has been identified in genetic analyses of major depressive disorder (MDD) and anxiety disorders (ADs)

  • Meta-analysis reveals WDR55 among genes with significant association signals (p=2.24×10⁻⁶)

  • WDR55 shows tissue-specific expression patterns relevant to psychiatric disorders in skin, blood, lung, and brain tissues

• Functional implications:

  • As a nucleolar protein involved in rRNA synthesis, WDR55 dysfunction could contribute to ribosomopathies

  • Cell cycle regulation defects associated with WDR55 might play roles in cancer biology

  • Its role in immune system development (thymus) suggests potential involvement in immunological disorders

The gene-level data demonstrates the complexity of WDR55's potential disease associations:

While these associations exist, further research is needed to establish causal relationships between WDR55 variants and specific human diseases .

How does WDR55 interact with other nucleolar proteins and what complexes does it form?

Unlike some other WD-repeat proteins involved in nucleolar function, WDR55 appears to operate through distinct molecular mechanisms:

• Protein interaction studies:

  • WDR55 was not co-immunoprecipitated with WDR12, Bop1, or Pes1, which form the PeBoW complex

  • This suggests WDR55 is a novel modulator of rRNA production that functions independently of the PeBoW complex

• Potential interaction partners:

  • As a WD40 repeat-containing protein, WDR55 likely serves as a scaffold for protein-protein interactions

  • Candidate interactors may include components of the rRNA processing machinery

  • In Arabidopsis, WDR55 interacts with DDB1A and likely forms a complex with CUL4, suggesting conserved interaction patterns

• Research approaches to identify interactors:

  • Proximity-based labeling methods (BioID, TurboID)

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • In silico structural prediction of interaction interfaces

Advanced structural biology techniques would be valuable for determining how WDR55's propeller-like structure mediates specific protein interactions relevant to its nucleolar functions.

What are the mechanistic differences between WDR55 function in plants versus animals?

Despite evolutionary divergence, WDR55 exhibits both conserved and distinct functions between plants and animals:

• Conserved functions:

  • Essential role in development across kingdoms

  • Involvement in cell cycle regulation

  • Interaction with DDB1/CUL4 complexes

• Kingdom-specific differences:

  • In plants (Arabidopsis), WDR55 affects apical symmetry establishment in embryos and influences leaf morphology and phyllotaxis

  • In animals (zebrafish/medaka), WDR55's developmental role focuses on thymus and swim bladder development

  • Plant WDR55 mutants can sometimes complete embryogenesis (weak alleles), while mouse WDR55 nulls are pre-implantation lethal

• Experimental approaches to study these differences:

  • Cross-species complementation experiments

  • Domain swapping between plant and animal WDR55 proteins

  • Comparative analysis of interacting partners

  • Examination of subcellular localization and trafficking

Research indicates that while the molecular function of WDR55 in rRNA processing may be conserved, its role in development has diverged to regulate kingdom-specific developmental processes, reflecting the different developmental programs of plants and animals .

What cutting-edge technologies can advance understanding of WDR55's role in ribosome biogenesis?

Several cutting-edge technologies can significantly advance our understanding of WDR55's role in ribosome biogenesis:

• Cryo-electron microscopy (cryo-EM):

  • Determine high-resolution structures of WDR55 within pre-ribosomal complexes

  • Visualize conformational changes during rRNA processing

  • Map the binding interface between WDR55 and rRNA or other processing factors

• Single-molecule RNA imaging:

  • Track rRNA processing in real-time in living cells

  • Visualize the dynamics of WDR55 association with pre-ribosomal particles

  • Quantify kinetic parameters of WDR55-dependent processing steps

• RNA modification analysis:

  • Investigate whether WDR55 influences specific rRNA modifications using nanopore direct RNA sequencing

  • Apply epitranscriptomic profiling to identify modification patterns affected by WDR55 dysfunction

• Integrative multi-omics approaches:

  • Combine proteomics, transcriptomics, and structural biology data

  • Create comprehensive models of WDR55's role in the nucleolar protein-RNA interaction network

  • Apply machine learning to predict functional consequences of WDR55 variants

• Genome-wide CRISPR screens:

  • Identify genetic interactions that exacerbate or suppress WDR55 deficiency phenotypes

  • Discover novel components of the WDR55-dependent rRNA processing pathway

These technologies would help resolve how WDR55 contributes to the accumulation of specific rRNA intermediates and clarify its distinct role separate from the PeBoW complex in ribosome biogenesis .

How suitable are zebrafish models for studying WDR55 function compared to other model organisms?

Zebrafish present several advantages and limitations for studying WDR55 function compared to other model organisms:

Advantages of zebrafish models:

• High genetic conservation (66% amino acid identity with medaka WDR55, 58-59% with human/mouse)

• Transparent embryos allowing direct visualization of developmental processes

• Rapid external development facilitating observation of phenotypes

• Well-established genetic manipulation tools (CRISPR/Cas9, transgenesis)

• Cost-effective compared to mammalian models

• Significant homology in developmental pathways with humans, sharing 84% of genes associated with human genetic diseases2

Limitations compared to other models:

• Less severe phenotypes than in mice (where WDR55-null is pre-implantation lethal)

• Some mammal-specific aspects of WDR55 function may not be captured

• Differences in immune system development compared to mammals

• Potential functional divergence in some pathways

Research strategy recommendations:

• Use zebrafish for initial developmental and cell biological studies

• Validate key findings in mammalian cell culture systems

• Reserve mouse models for specific aspects requiring mammalian context

• Consider Arabidopsis for evolutionary comparative studies of WDR55 function

The zebrafish model offers particular advantages for studying WDR55's role in thymus development, as thymus defects are readily observable and have been well-characterized in both zebrafish and medaka WDR55 mutants .

What protocols should be followed for expression and purification of recombinant WDR55 for structural studies?

For structural studies of recombinant WDR55, optimization of expression and purification is critical:

• Expression systems comparison:

  • Yeast expression systems have been successfully used for recombinant Danio rerio WDR55 production

  • E. coli systems may be suitable but require optimization due to potential folding issues

  • Mammalian cell expression (HEK-293) could provide proper post-translational modifications

  • Baculovirus/insect cell systems might balance yield and proper folding

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged WDR55

  • Size exclusion chromatography to ensure monodispersity

  • Ion exchange chromatography for further purification

  • Consider on-column refolding if inclusion bodies form

• Buffer optimization for structural studies:

  • Screen various pH conditions (typically pH 7-8)

  • Test different salt concentrations (100-300 mM NaCl range)

  • Add stabilizing agents (5-10% glycerol, low concentrations of reducing agents)

  • Include protease inhibitors to prevent degradation

• Quality control assessments:

  • SDS-PAGE for purity (target >95% for structural studies)

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to identify stabilizing conditions

  • Limited proteolysis to identify stable domains

For crystallography or cryo-EM studies, it may be beneficial to remove flexible regions (particularly the C-terminal unstructured region) while maintaining the core WD40 repeat domains that form the characteristic propeller structure .

What are promising research directions for understanding WDR55's role in human disease pathways?

Several promising research directions could further elucidate WDR55's role in human disease pathways:

• Psychiatric disorder connections:

  • Investigate the mechanistic link between WDR55 variants identified in depression/anxiety studies and neuronal function

  • Examine how WDR55-mediated ribosome biogenesis affects neuron-specific translation in relevant brain regions

  • Explore WDR55's potential role in stress response pathways common to psychiatric conditions

• Cancer biology:

  • Analyze WDR55 expression across cancer types, particularly those with nucleolar dysfunction

  • Investigate whether WDR55 alterations contribute to dysregulated cell cycle progression in cancer cells

  • Explore the therapeutic potential of targeting WDR55-dependent pathways in cancer

• Developmental disorders:

  • Screen for WDR55 variants in patients with congenital disorders of unknown etiology

  • Investigate WDR55's role in thymus development and potential connections to immune deficiencies

  • Create patient-specific induced pluripotent stem cell models to study developmental impacts of WDR55 variants

• Integration with established disease pathways:

  • Explore connections between WDR55 and the p53 pathway in disease contexts

  • Investigate potential relationships between WDR55 and established ribosomopathies

  • Examine whether WDR55 interacts with disease-associated nucleolar proteins

These research directions would benefit from integrating zebrafish models with human genetic data and patient-derived cellular models to establish causality between WDR55 dysfunction and disease phenotypes.

How might WDR55 function be targeted therapeutically in relevant disease conditions?

Potential therapeutic approaches targeting WDR55 function could include:

• Small molecule modulators:

  • Screen for compounds that restore proper localization of mutant WDR55 proteins

  • Identify molecules that normalize aberrant rRNA processing caused by WDR55 dysfunction

  • Develop drugs that modulate WDR55 protein-protein interactions in disease contexts

• Gene therapy approaches:

  • Develop CRISPR-based strategies to correct pathogenic WDR55 variants

  • Utilize AAV vectors for delivery of functional WDR55 in affected tissues

  • Explore RNA-based therapeutics to modulate WDR55 expression levels

• Pathway-based interventions:

  • Target downstream effects of WDR55 dysfunction (e.g., p53 pathway activation)

  • Develop strategies to mitigate nucleolar stress responses

  • Identify compensatory pathways that could bypass WDR55 requirements

• Drug repurposing opportunities:

  • Evaluate existing nucleolar-targeting compounds for effects on WDR55-dependent processes

  • Investigate whether approved cell cycle regulators could normalize defects caused by WDR55 dysfunction

  • Screen FDA-approved drugs for those that restore normal rRNA processing in WDR55-deficient cells

Development of such therapies would require detailed understanding of tissue-specific WDR55 functions and careful consideration of potential off-target effects, given WDR55's fundamental role in ribosome biogenesis and cell cycle regulation.