Recombinant Danio rerio UPF0568 protein C14orf166 homolog (zgc:56576)

What is the C14orf166 protein in Danio rerio and how does it compare to human orthologues?

The C14orf166 protein in Danio rerio (zebrafish) is a 242-amino acid protein also known as UPF0568 protein C14orf166 homolog or zgc:56576. It functions as an RNA transcription, translation, and transport factor protein. The zebrafish C14orf166 shares significant homology with human C14orf166 (also known as RTRAF), which encodes a 28-kDa nuclear and cytoplasmic protein involved in viral infection, RNA metabolism, and centrosome structure. Comparative analysis reveals conservation of key functional domains across vertebrate species, making zebrafish an appropriate model for studying the protein's fundamental biological roles. The zebrafish protein maintains the characteristic sequence motifs necessary for its interaction with RNA processing machinery, suggesting evolutionary conservation of its core functions in RNA metabolism.

What expression systems are available for producing recombinant Danio rerio C14orf166?

Multiple expression systems can be utilized for producing recombinant Danio rerio C14orf166, each with distinct advantages depending on research requirements:

The yeast protein expression system represents a balance between the economical benefits of prokaryotic systems and the proper protein folding capabilities of eukaryotic systems. For zebrafish C14orf166, yeast expression can provide proteins with modifications such as glycosylation, acylation, and phosphorylation to ensure native protein conformation. This makes it particularly suitable for producing research-grade material that closely resembles the natural protein.

How can researchers verify the purity and identity of recombinant Danio rerio C14orf166?

Verification of recombinant Danio rerio C14orf166 purity and identity requires a multi-method analytical approach:

• SDS-PAGE analysis: Standard method for assessing protein purity. Commercial preparations typically achieve >90% purity as determined by SDS-PAGE.

• Western blot confirmation: Using specific antibodies against the His-tag or against C14orf166 epitopes to confirm protein identity.

• Functional assays: Verification of biological activity through RNA binding assays or interaction studies with known binding partners (e.g., RNA polymerase II components).

This comprehensive verification ensures that experimental results using the recombinant protein can be attributed to C14orf166 activity rather than contaminants or improperly folded protein.

What are the key functional domains of C14orf166 in Danio rerio and how do they contribute to its biological activity?

The C14orf166 protein in Danio rerio contains several functionally significant domains that are critical for its biological activities:

• RNA-binding domain: Located in the N-terminal region, this domain facilitates interaction with various RNA species, enabling the protein's role in RNA processing and metabolism.

• Nuclear localization signal (NLS): This sequence directs the protein to the nucleus, allowing it to participate in transcriptional regulation and RNA processing.

• Protein-interaction domains: These regions mediate binding to key partners including DDX1, HSPC117, and FAM98B to form functional complexes involved in RNA metabolism.

• C-terminal regulatory region: Contains phosphorylation sites that may modulate protein activity in response to cellular signaling.

These domains work cooperatively to enable C14orf166's multifunctional role in RNA metabolism, transcriptional regulation, and viral replication processes. Structural studies suggest that the protein's tertiary structure creates surface pockets that accommodate both nucleic acid and protein binding, explaining its versatility in forming different functional complexes depending on cellular context.

What protein complexes does C14orf166 form in Danio rerio and what are their functions?

In Danio rerio, as in other vertebrates, C14orf166 forms several functionally distinct protein complexes:

The C14orf166-DDX1-HSPC117-FAM98B complex plays a crucial role in regulating RNA metabolism and determining RNA fate. In zebrafish development, this complex may be particularly important for proper embryonic development and organogenesis, as RNA metabolism is tightly regulated during these processes. The interaction with RNA Polymerase II indicates involvement in transcriptional control, while centrosomal localization suggests functions in cell division. These multiple roles make C14orf166 a versatile regulator of fundamental cellular processes in vertebrate development.

How does C14orf166 participate in RNA metabolism in zebrafish models?

C14orf166 participates in multiple aspects of RNA metabolism in zebrafish models, functioning as a key regulatory molecule throughout the RNA lifecycle:

• Transcriptional regulation: C14orf166 associates with RNA polymerase II to influence transcription initiation. In zebrafish models, this activity may regulate the expression of developmentally important genes.

• RNA processing: The protein contributes to post-transcriptional modifications, including splicing and polyadenylation, which are critical for proper gene expression during zebrafish development.

• RNA transport: As part of the C14orf166-DDX1-HSPC117-FAM98B complex, it facilitates the movement of RNA between cellular compartments, ensuring proper spatial distribution of transcripts.

• RNA stability: Evidence suggests that C14orf166 influences the half-life of certain RNA species by modulating their susceptibility to degradation pathways.

In zebrafish development, these functions may be particularly important during early embryogenesis when precise control of maternal RNA stability and zygotic gene activation is essential. Researchers studying developmental processes in zebrafish can utilize recombinant C14orf166 in RNA immunoprecipitation experiments to identify target RNAs regulated by this protein during different developmental stages.

What are the recommended protocols for using recombinant Danio rerio C14orf166 in ELISA assays?

For optimal results in ELISA applications using recombinant Danio rerio C14orf166 (His-tagged), researchers should follow this methodology:

• Coating: Dilute recombinant C14orf166 protein to 1-10 μg/mL in carbonate buffer (pH 9.6). Add 100 μL per well to high-binding ELISA plates and incubate overnight at 4°C.

• Blocking: Wash wells 3 times with PBST (PBS + 0.05% Tween-20), then block with 300 μL of blocking buffer (PBS + 1-5% BSA or non-fat milk) for 1-2 hours at room temperature.

• Primary antibody: Add diluted anti-C14orf166 antibodies or test sera and incubate for 1-2 hours at room temperature or overnight at 4°C.

• Detection: After washing, add appropriate HRP-conjugated secondary antibody and incubate for 1 hour. Develop with TMB substrate and read absorbance at 450 nm.

• Controls and validation:

  • Positive control: Known anti-C14orf166 antibody

  • Negative control: Wells without primary antibody

  • Specificity control: Wells coated with irrelevant His-tagged protein

This protocol is particularly useful for validation of antibodies against C14orf166, protein-protein interaction studies, and detection of anti-C14orf166 antibodies in experimental samples. The high purity (>90%) of commercially available recombinant C14orf166 ensures reliable results when following this methodology.

How can researchers design co-immunoprecipitation experiments to study C14orf166 interaction partners in zebrafish models?

Designing effective co-immunoprecipitation (co-IP) experiments to investigate C14orf166 interaction partners in zebrafish models requires careful planning:

• Sample preparation:

  • Homogenize zebrafish embryos or adult tissues in non-denaturing lysis buffer (typically containing 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% NP-40, protease inhibitors)

  • Incubate lysate on ice for 30 minutes, then centrifuge at 13,000×g for 15 minutes at 4°C to remove debris

  • Pre-clear lysate with protein A/G beads to minimize non-specific binding

• Immunoprecipitation:

  • Option A (endogenous proteins): Incubate lysate with specific anti-C14orf166 antibody

  • Option B (tagged recombinant protein): Use anti-tag antibody (e.g., anti-His antibody) or affinity resin

  • Add protein A/G beads and incubate with rotation overnight at 4°C

  • Wash beads thoroughly (at least 4 times) with wash buffer

• Analysis:

  • Elute bound proteins with SDS sample buffer and separate by SDS-PAGE

  • Analyze by Western blot to detect specific interaction partners (DDX1, HSPC117, FAM98B)

  • For discovery of novel partners, perform mass spectrometry analysis

• Controls:

  • Negative control: IgG from same species as primary antibody

  • Input control: Analyze a portion of pre-immunoprecipitation lysate

  • Validation through reverse co-IP: Immunoprecipitate suspected partner and probe for C14orf166

This methodology allows for robust identification of physiologically relevant protein interactions in the zebrafish model, particularly important for developmental studies where C14orf166 complex composition may change during different stages.

What RNA-protein interaction assays are most effective for studying C14orf166 function in zebrafish?

Several RNA-protein interaction assays are particularly effective for investigating C14orf166 function in zebrafish systems:

When implementing these assays with zebrafish samples, researchers should consider developmental stage-specific effects, as C14orf166 may regulate different RNA targets during embryogenesis versus adult tissues. The recombinant Danio rerio C14orf166 protein can serve as a valuable positive control in these experiments, particularly for establishing binding specificity in EMSA and RNA pull-down assays. Additionally, comparing RNA targets between zebrafish and mammalian systems can provide evolutionary insights into conserved regulatory networks.

How does the role of C14orf166 in viral infection mechanisms translate from human studies to zebrafish models?

Translating C14orf166's viral response functions from human studies to zebrafish models presents both opportunities and challenges:

• Conservation of interaction mechanisms: The zebrafish C14orf166 homolog shows significant sequence similarity in domains that mediate viral protein interactions in humans. In human studies, C14orf166 binds to the polymerase acidic protein subunit of influenza A virus and interacts with hepatitis C virus core protein. Zebrafish models can be used to determine whether these interaction interfaces are conserved across species.

• Experimental methodology for viral studies in zebrafish:

  • Generation of transgenic zebrafish expressing tagged C14orf166 for visualization during infection

  • Infection protocols using zebrafish-compatible viruses (e.g., viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus)

  • Comparison of wild-type and C14orf166-knockdown zebrafish response to viral challenge

  • Protein-protein interaction studies using recombinant zebrafish C14orf166 and viral proteins

• Comparative functional analysis: Although zebrafish lack direct counterparts to human influenza and hepatitis viruses, the mechanisms by which C14orf166 interacts with viral machinery may be conserved. Researchers can investigate whether zebrafish C14orf166 participates in general antiviral responses or influences expression of interferon-stimulated genes.

• Data interpretation framework:

This research direction provides valuable insights into the evolutionary conservation of host-pathogen interactions and may identify new targets for broad-spectrum antiviral interventions.

What are the methodological considerations for CRISPR/Cas9-mediated functional studies of C14orf166 in zebrafish?

CRISPR/Cas9-mediated functional studies of C14orf166 in zebrafish require careful consideration of several methodological factors:

• Guide RNA design strategy:

  • Target early exons to ensure complete loss of function

  • Avoid regions with high homology to other genes

  • Design multiple gRNAs (minimum 3-4) targeting different regions

  • Recommended target sites: exons encoding RNA-binding domain and protein interaction domains

  • Evaluate potential off-targets using zebrafish genome databases

• Delivery method optimization:

  • Microinjection of Cas9 protein (instead of mRNA) with gRNAs into one-cell stage embryos

  • Recommended concentrations: 300-500 ng/μL Cas9 protein with 25-50 ng/μL gRNA

  • Include phenol red (0.05%) for visualization during injection

• Validation strategies:

  • Primary screening: T7 endonuclease assay or heteroduplex mobility assay

  • Secondary confirmation: Sanger sequencing of targeted regions

  • Protein level verification: Western blot using antibodies against zebrafish C14orf166

• Addressing potential challenges:

  • Early lethality: Generate conditional knockouts if complete knockout is lethal

  • Functional compensation: Screen for upregulation of paralogous genes

  • Mosaicism: Establish F2 generation for homogeneous mutant population

• Phenotypic analysis framework:

  • Developmental timeline assessment with standardized staging

  • RNA metabolism evaluation using transcriptome analysis

  • Cellular localization studies of RNA processing factors

  • Challenge experiments to assess response to stressors or viral infection

Using recombinant Danio rerio C14orf166 protein for rescue experiments provides an essential control to confirm phenotype specificity. Researchers should also consider generating epitope-tagged knockin lines for in vivo protein localization and chromatin immunoprecipitation studies.

How can researchers investigate the role of C14orf166 in zebrafish embryonic development through temporal expression profiling?

Investigating C14orf166's role in zebrafish embryonic development through temporal expression profiling requires a multi-dimensional approach:

• Quantitative expression analysis across developmental stages:

  • Collect embryos at key developmental timepoints: 0 hpf (hours post-fertilization), 3 hpf, 6 hpf, 12 hpf, 24 hpf, 48 hpf, 72 hpf, and 5 dpf

  • Extract total RNA using TRIzol or equivalent reagent

  • Perform RT-qPCR targeting C14orf166 mRNA with stage-appropriate reference genes (ef1α, β-actin)

  • Create expression timeline normalized to reference genes

• Spatial expression pattern determination:

  • Whole-mount in situ hybridization (WISH) using antisense RNA probes targeting C14orf166

  • Immunohistochemistry using specific antibodies against zebrafish C14orf166

  • Tissue-specific analysis through laser-capture microdissection followed by RT-qPCR

• Functional correlation analysis:

  • Compare expression patterns with key developmental events

  • Cross-reference with expression of known interaction partners (DDX1, HSPC117, FAM98B)

  • Temporal correlation with activation of zygotic genome transcription

• Manipulation studies:

  • Morpholino-mediated knockdown at specific developmental stages

  • Heat-shock inducible transgenic overexpression

  • CRISPR interference for temporal-specific gene silencing

• Multi-omics integration:

  • RNA-seq to identify transcriptome changes corresponding to C14orf166 expression fluctuations

  • RIP-seq to identify stage-specific RNA targets

  • Proteomics to determine changing interaction partners during development

This comprehensive profiling approach enables researchers to construct a detailed model of C14orf166 function throughout zebrafish development, providing insights into its evolutionary conserved roles in vertebrate embryogenesis.

How can zebrafish C14orf166 models contribute to understanding its role in human cancer pathogenesis?

Zebrafish C14orf166 models offer unique advantages for understanding the protein's role in human cancer pathogenesis:

• Translational research framework:

  • Human studies show C14orf166 overexpression in multiple cancer types

  • Zebrafish models allow in vivo visualization of cancer-related processes

  • Conservation of key signaling pathways (GSK3β pathway, IL-6/STAT3) between humans and zebrafish

• Model development strategies:

  • Transgenic overexpression under tissue-specific promoters to mimic cancer overexpression

  • CRISPR/Cas9 engineering of specific mutations identified in human tumors

  • Xenograft models using human cancer cells in zebrafish embryos to study C14orf166 function

• Mechanistic investigations:

  • Real-time imaging of C14orf166-overexpressing cells during proliferation, migration, and invasion

  • Assessment of impact on glycogen synthase kinase 3β-mediated signaling, which is implicated in human cancers

  • Evaluation of effects on retinoblastoma protein regulation and cell cycle control

  • Analysis of IL-6 expression and STAT3 activation in response to altered C14orf166 levels

• Comparative pathway analysis:

• Drug screening applications:

  • High-throughput screening of compounds targeting C14orf166 or its downstream pathways

  • Rapid assessment of in vivo efficacy in cancer models

  • Evaluation of specificity through rescue experiments with recombinant protein

These zebrafish models provide a powerful complement to mammalian systems for understanding C14orf166's oncogenic mechanisms, potentially leading to new therapeutic approaches for cancers where this protein is dysregulated.

What experimental approaches can investigate the evolutionary conservation of C14orf166 functions between zebrafish and mammals?

Investigating evolutionary conservation of C14orf166 functions between zebrafish and mammals requires systematic comparative approaches:

• Sequence and structural homology analysis:

  • Multiple sequence alignment of C14orf166 protein sequences across species

  • Homology modeling of zebrafish C14orf166 based on mammalian structures

  • Conservation mapping of functional domains and interaction interfaces

  • Phylogenetic analysis to determine evolutionary relationships

• Cross-species functional complementation:

  • Rescue experiments in C14orf166-deficient zebrafish using mammalian orthologs

  • Expression of zebrafish C14orf166 in mammalian cell lines with CRISPR knockout

  • Domain-swapping experiments to identify species-specific functional regions

• Comparative interactome analysis:

  • Parallel co-IP experiments in zebrafish and mammalian systems

  • Cross-species protein-protein interaction studies using recombinant proteins

  • Identification of conserved vs. species-specific interaction partners

• Pathway conservation assessment:

  • Comparative transcriptomics after C14orf166 depletion in fish and mammalian cells

  • Analysis of effects on conserved signaling pathways (RNA processing, centrosome function)

  • Cross-species chromatin immunoprecipitation to identify conserved genomic targets

• Systematic data integration framework:

These comparative approaches provide insights into the core ancestral functions of C14orf166 that have been maintained through vertebrate evolution, as well as potential species-specific adaptations. The high purity recombinant proteins available from both zebrafish and mammalian sources facilitate direct biochemical comparison studies.

How can researchers design experiments to evaluate C14orf166's role in zebrafish immune response to viral challenges?

Designing experiments to evaluate C14orf166's role in zebrafish immune response to viral challenges requires an integrated approach:

• Viral challenge model development:

  • Select appropriate zebrafish-infecting viruses (SVCV, IHNV, or VHSV)

  • Establish infection protocols (immersion, injection) and viral dose standardization

  • Determine infection kinetics through viral load quantification

  • Set up survival curves for wild-type zebrafish as baseline

• C14orf166 manipulation strategies:

  • Generate stable C14orf166 knockout or knockdown lines

  • Create tissue-specific conditional knockouts focusing on immune tissues

  • Develop transgenic overexpression lines with either constitutive or inducible promoters

  • Establish rescue lines using recombinant C14orf166 protein or mRNA injection

• Comprehensive immune response assessment:

  • Quantify viral replication kinetics via RT-qPCR or plaque assays

  • Assess innate immune response through expression analysis of interferons and interferon-stimulated genes

  • Evaluate inflammatory cytokine profiles (IL-1β, TNF-α, IL-6)

  • Analyze leukocyte recruitment and activation in infected tissues

• Molecular mechanism elucidation:

  • Investigate C14orf166 localization changes during infection

  • Perform immunoprecipitation to identify viral components interacting with C14orf166

  • Conduct RNA-seq to identify virus-induced transcriptome changes dependent on C14orf166

  • Assess impact on antiviral signaling pathways (RIG-I-like receptors, interferon signaling)

• Translation to therapeutic insights:

This experimental framework provides a comprehensive assessment of C14orf166's role in antiviral immunity in zebrafish, with potential implications for understanding similar mechanisms in humans. The high-quality recombinant protein enables precise mechanistic studies of direct interactions with viral components and immune signaling molecules.

What are the key methodological considerations when integrating findings from zebrafish C14orf166 studies into broader vertebrate biology?

Integrating findings from zebrafish C14orf166 studies into broader vertebrate biology requires careful methodological considerations to ensure valid cross-species extrapolation. Researchers should establish clear homology relationships through phylogenetic analysis and sequence comparison, using quantitative metrics to assess functional domain conservation. When experimental findings from zebrafish differ from mammalian models, these differences should be systematically investigated through complementation studies. The integration process should acknowledge both the strengths of the zebrafish model (visualization of developmental processes, genetic tractability) and its limitations (potential divergence in specific molecular pathways). Ultimately, constructing a multi-species molecular network around C14orf166 provides the most comprehensive understanding of its conserved functions in RNA metabolism, transcriptional regulation, and response to viral infection across vertebrate evolution.