Recombinant Danio rerio Protein pelota homolog (pelo)

What is the pelota homolog (pelo) in zebrafish and what are its basic genetic characteristics?

Pelota homolog (pelo) in Danio rerio is characterized as an mRNA surveillance and ribosome rescue factor. The gene is officially designated with the symbol "pelo" and is identified with the following molecular details:

The pelo gene produces a protein that plays essential roles in translation quality control and ribosome recycling, similar to its homologs in other species .

How does pelo function in normal zebrafish development?

Pelo functions as part of the mRNA surveillance pathway during zebrafish development, ensuring the quality control of protein synthesis. The protein is involved in ribosome rescue when translation is stalled, helping maintain proper protein expression during critical developmental stages. Studies using zebrafish as a model organism have shown that proper gene expression regulation is essential for development, particularly in rapidly dividing embryonic tissues where translation fidelity is crucial .

Zebrafish embryos, with their rapid and externally observable development, provide an excellent system to study pelo function through their transparent nature, allowing visualization of developmental processes in real-time. Expression analysis typically shows pelo activity in tissues with high protein synthesis demands, similar to other translation-associated factors .

What conservation patterns does pelo show across vertebrate species?

Pelo demonstrates significant evolutionary conservation across vertebrate species, reflecting its fundamental role in mRNA surveillance and translation. The conservation pattern includes:

This high conservation makes zebrafish pelo an excellent proxy for studying mechanisms that might be relevant to human disease, as fundamental translation quality control mechanisms are maintained across vertebrates .

What are optimal methods for gene knockout or knockdown of pelo in zebrafish?

Several methodologies can be employed for pelo gene manipulation in zebrafish, each with specific advantages depending on research objectives:

CRISPR/Cas9 Gene Editing (Complete Knockout):

• Design guide RNAs targeting exons 9-10 of pelo based on the reference sequence NM_201136.1

• Inject CRISPR/Cas9 complexes into one-cell stage embryos

• Screen F0 mosaic fish via fin clip genotyping using PCR amplification across the target site

• Establish stable F1 lines through outcrossing and genotyping

Morpholino Antisense Oligonucleotides (Transient Knockdown):

• Design splice-blocking morpholinos targeting exon 9-10 junction

• Inject 1-4 ng morpholino into 1-2 cell stage embryos

• Validate knockdown efficiency via RT-PCR to detect altered splicing products

• Include control morpholino injections to distinguish specific phenotypes

Dominant Negative Approach:

• Clone truncated pelo coding sequence into expression vector with tissue-specific promoter

• Generate stable transgenic lines using Tol2 transposon system

• Induce expression at desired developmental stages

• Validate transgene expression via fluorescent reporter co-expression

When designing knockout experiments, researchers should consider that complete pelo deletion might cause embryonic lethality based on its crucial role in translation, so conditional knockout approaches may be preferable .

How can I design primers for pelo gene expression analysis in zebrafish?

For robust pelo expression analysis in zebrafish, primer design should consider the following guidelines:

• qRT-PCR Primer Design:

  • Target exon-exon junctions, particularly around exons 9-10 (assay location 987) to prevent genomic DNA amplification

  • Design primers that generate a short amplicon (optimal range: 50-150 bp)

  • Example primers based on NM_201136.1:

    • Forward: 5'-CAGATCTGCAAGGTGTGGAAG-3' (exon 9)

    • Reverse: 5'-GTCTGCAGTCTGAGGTCTGAA-3' (exon 10)

    • Expected amplicon: 58 bp

• Reference Gene Selection:

  • Include at least 3 reference genes for normalization (e.g., ef1α, rpl13a, and actb1)

  • Validate reference gene stability under experimental conditions using algorithms like geNorm or NormFinder

• Expression Analysis Methodology:

  • For developmental studies: Sample at key developmental stages (e.g., 6, 24, 48, 72 hpf)

  • For tissue-specific analysis: Isolate RNA from dissected tissues (brain, muscle, liver, etc.)

  • Standard reaction conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15s and 60°C for 1 min

When analyzing pelo expression in response to experimental manipulations, it's essential to monitor both mRNA levels and protein expression, as post-transcriptional regulation can affect final protein abundance.

What phenotypes would be expected in pelo-deficient zebrafish?

Based on the role of pelo in mRNA surveillance and ribosome rescue, the following phenotypes might be expected in pelo-deficient zebrafish:

Phenotype assessment should include:

• Morphological analysis during development (body axis, organ formation)

• Behavioral assays to detect neurological defects

• Cell death analysis using acridine orange staining

• Tissue-specific analysis focusing on high-protein turnover tissues

Since pelo plays a role in mRNA surveillance, researchers should also consider monitoring stress response markers and immune function, as zebrafish have demonstrated capability for mounting immune responses to various challenges .

How does pelo function in zebrafish immune responses?

Pelo's role in zebrafish immune responses is multifaceted due to its fundamental function in translation quality control, which affects immune cell development and function:

• Immune Cell Development:

  • Pelo likely influences hematopoiesis and immune cell differentiation through its role in protein synthesis quality control

  • Zebrafish hematopoietic tissues (kidney marrow equivalent) would show altered profiles in pelo-deficient models

• Antiviral Response Modulation:

  • Studies in zebrafish have demonstrated robust antiviral immune responses, with upregulation of TLR3, IFNαβ, Mx, IFNγ and TNFα expression at 72h post-infection in kidney tissues during viral challenges

  • Pelo may contribute to the regulation of these antiviral response genes through its role in translation regulation and mRNA surveillance

• Experimental Approaches to Study pelo in Immune Context:

  • Challenge studies using VHSV (viral hemorrhagic septicemia virus) can evaluate how pelo affects immune response efficacy

  • Analysis of expression of immune genes like TLR3, IFNαβ, Mx, IFNγ and TNFα in pelo-deficient versus wild-type fish would provide insights into pelo's role in immune modulation

  • Flow cytometry analysis of kidney marrow cells to evaluate changes in immune cell populations

Zebrafish models are particularly valuable for such studies as they can mount effective antiviral immune responses even at relatively low temperatures (15°C), demonstrating the functional conservation of immune mechanisms between fish and mammals .

What methods are optimal for recombinant expression and purification of zebrafish pelo protein?

For successful recombinant expression and purification of zebrafish pelo protein, researchers should consider the following methodological approaches:

Expression System Selection:

• E. coli-based expression:

  • Clone the pelo coding sequence (based on NM_201136.1) into pET vectors with appropriate tags (6xHis or GST)

  • Express in BL21(DE3) or Rosetta strains to account for codon bias

  • Induce at low temperature (16-18°C) to enhance solubility

  • Typical yields: 2-5 mg/L culture

• Insect cell expression:

  • Clone pelo into baculovirus vectors (pFastBac)

  • Express in Sf9 or High Five cells

  • Harvest 72-96 hours post-infection

  • Typical yields: 5-10 mg/L culture

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM DTT

• Affinity chromatography using Ni-NTA for His-tagged protein or glutathione resin for GST-tagged constructs

• Size exclusion chromatography using Superdex 75 or 200 columns

• Quality assessment via SDS-PAGE and Western blotting

Activity Assays:

• RNA binding assays using fluorescence anisotropy

• ATPase activity measurements

• In vitro translation termination assays with purified ribosomal components

Recombinant pelo protein can be used for structural studies, interaction analyses, and in vitro biochemical assays to understand its molecular function in mRNA surveillance pathways .

How can transgenic zebrafish models be optimized for studying pelo function?

Transgenic zebrafish models offer powerful approaches for studying pelo function in vivo. Optimized strategies include:

Conditional Expression Systems:

• Gal4/UAS System:

  • Generate fish expressing Gal4 under tissue-specific promoters

  • Create responder lines with UAS:pelo-wildtype and UAS:pelo-mutant constructs

  • Cross driver and responder lines to achieve tissue-specific expression

  • Monitor expression using fluorescent protein fusions (e.g., mCherry)

• Heat-shock Inducible System:

  • Clone pelo variants under hsp70 promoter control

  • Achieve temporal control of expression through controlled temperature shifts

  • This approach has been validated in zebrafish enhancer studies with precise control

Integration Methods:

• Tol2 Transposon System:

  • Enables relatively efficient genomic integration

  • May result in multiple insertion sites and position effects

  • Suitable for rapid generation of transgenic lines

• PhiC31 Integrase System:

  • Allows site-directed integration at predefined genomic loci

  • Reduces position effect variation commonly found in transposon-based assays

  • Provides more consistent expression levels across independent lines

  • Has been validated for enhancer testing in zebrafish

Analytical Approaches:

• Tissue-specific transcriptomics to identify pelo-dependent gene expression

• Ribosome profiling to detect translation defects

• Live imaging of tagged pelo to monitor subcellular localization and dynamics

When creating transgenic models, researchers should consider potential developmental effects of pelo overexpression or dominant-negative variants, which might require careful titration of expression levels or restricting expression to specific developmental timepoints .

How can zebrafish pelo models contribute to understanding human disease mechanisms?

Zebrafish pelo models provide valuable insights into human disease mechanisms through several research approaches:

• Modeling Translation-Related Disorders:

  • Neurodegenerative diseases often involve defects in protein quality control

  • Pelo-deficient zebrafish can model aspects of these disorders through impaired mRNA surveillance

  • Study outcomes include behavioral phenotyping, histological analysis, and molecular profiling

• Comparative Genomics Approach:

  • Human PELO gene mutations can be recreated in zebrafish pelo

  • Phenotypic analysis of these mutations in zebrafish provides functional validation of human variants

  • Example workflow:

    • Identify human PELO variants of interest

    • Generate equivalent mutations in zebrafish pelo

    • Analyze phenotypes at organismal, cellular, and molecular levels

• Drug Discovery Applications:

  • Screen compounds that might rescue pelo-deficient phenotypes

  • Assess impacts on translation quality control pathways

  • Validate hits through secondary assays and dose-response studies

Zebrafish offers significant advantages for these studies through its combination of vertebrate biology, genetic tractability, and experimental accessibility. The high fecundity and external fertilization facilitate large-scale studies while the transparent embryo enables direct visualization of developmental and pathological processes .

What are the technical challenges in generating CRISPR/Cas9 pelo knockout zebrafish lines?

Generating CRISPR/Cas9 pelo knockout zebrafish lines presents several technical challenges that researchers should anticipate:

• Guide RNA Design Considerations:

  • Target exons 9-10 for maximum disruption based on reference sequence NM_201136.1

  • Avoid off-target effects by selecting guides with minimal predicted off-target sites

  • Recommended tools: CHOPCHOP, CRISPRscan, and CRISPOR for guide design

• Mosaicism Management:

  • F0 fish typically show mosaic genotypes due to CRISPR activity after the first cell division

  • Strategy: Raise multiple F0 founders and screen for germline transmission

  • Genotyping protocol: Extract DNA from F1 embryos or adult fin clips, perform PCR across target site, and sequence to identify mutations

• Phenotyping Challenges:

  • Maternal contribution of pelo mRNA may mask early phenotypes in homozygous mutants

  • Solution: Generate maternal-zygotic mutants by incrossing homozygous adults

  • Phenotypic analysis should span multiple developmental stages (24 hpf, 48 hpf, 72 hpf, and beyond)

• Validation Requirements:

  • Confirm knockout at protein level using Western blot or immunohistochemistry

  • Generate rescue lines by expressing wild-type pelo to confirm phenotype specificity

  • Create multiple independent alleles to distinguish specific phenotypes from off-target effects

The zebrafish model's ability to rapidly generate mutant lines through CRISPR technology, combined with its vertebrate physiology, makes it an excellent system for studying pelo function despite these challenges .

How do temperature conditions affect pelo function in zebrafish research models?

Temperature conditions significantly impact zebrafish physiology and pelo function, presenting important considerations for research design:

• Temperature Range Considerations:

  • Standard zebrafish laboratory conditions: 26-28°C

  • Temperature tolerance range in wild zebrafish: 24.6–38.6°C

  • Lower temperature experiments (15-23°C): Slow development but potentially useful for certain studies

  • Higher temperature experiments (30-33°C): Accelerated development but increased stress

• Pelo Function and Temperature Relationship:

  • Translation quality control mechanisms may be differentially regulated at various temperatures

  • At lower temperatures (e.g., 15°C), zebrafish can still mount immune responses, suggesting functional translation machinery including pelo-mediated quality control

  • Experimental evidence shows that zebrafish can mount efficient antiviral immune responses at 15°C, indicating functional translation quality control mechanisms at lower temperatures

• Experimental Design Implications:

  • For immune studies: Consider that zebrafish can be maintained at 15°C while studying immune responses

  • For developmental studies: Standard 28°C provides optimal development timing

  • For stress response studies: Heat shock at 37°C can be used to study pelo's role under proteotoxic stress conditions

• Temperature Shift Protocols:

  • Gradual temperature changes (1°C/hour) minimize stress responses

  • For heat shock experiments: 37°C for 1 hour followed by recovery at 28°C

  • For cold shock experiments: Gradually reduce to target temperature over 2-3 hours

Temperature manipulation can be used as an experimental variable when studying pelo function, particularly in the context of stress responses and protein quality control mechanisms. The broad temperature tolerance of zebrafish makes it a versatile model for studying temperature-dependent aspects of pelo function .

How can pelo function in zebrafish inform research on mRNA surveillance in human disease?

Zebrafish pelo studies provide valuable insights into human mRNA surveillance mechanisms with significant disease implications:

• Conservation of Surveillance Mechanisms:

  • Pelo-mediated mRNA surveillance pathways are highly conserved between zebrafish and humans

  • Studies in zebrafish can reveal fundamental mechanisms relevant to human translation quality control

  • Research findings in zebrafish models can be extrapolated to human disease contexts with appropriate validation

• Disease Modeling Applications:

  • Neurodegenerative diseases: Many involve defects in protein quality control and RNA processing

  • Cancer biology: Dysregulated translation is a hallmark of many cancers

  • Developmental disorders: Mutations in translation factors cause numerous congenital conditions

• Methodological Approach for Translational Research:

  • Generate zebrafish models with pelo mutations corresponding to human disease variants

  • Conduct high-throughput drug screens using pelo mutant phenotypes as readouts

  • Validate findings through complementary mammalian cell culture experiments

• Future Research Directions:

  • Integrate zebrafish pelo studies with human patient-derived cells

  • Develop tissue-specific pelo knockout models to understand organ-specific requirements

  • Apply advanced imaging techniques to visualize pelo function in real-time in living organisms

The zebrafish model offers unique advantages through its combination of genetic tractability, vertebrate physiology, and experimental accessibility, positioning it as an ideal bridge between simpler model organisms and mammalian systems for studying pelo function in human disease contexts .

What cutting-edge techniques are emerging for studying pelo protein interactions in zebrafish?

Several cutting-edge techniques are revolutionizing the study of pelo protein interactions in zebrafish:

• Proximity Labeling Approaches:

  • BioID or TurboID fusion with pelo to identify proximal interacting proteins in vivo

  • Workflow: Generate transgenic lines expressing pelo-BioID, administer biotin, isolate biotinylated proteins, and identify by mass spectrometry

  • Advantages: Captures transient interactions and functions in native cellular context

• CRISPR-Based Tagging Strategies:

  • CRISPR knock-in of fluorescent or affinity tags at the endogenous pelo locus

  • Example protocol: Design homology-directed repair templates with mNeonGreen or 3xFLAG tags

  • Advantages: Preserves endogenous regulation and expression levels

• Single-Cell Approaches:

  • Single-cell RNA-seq to identify cell type-specific responses to pelo disruption

  • Single-cell ribosome profiling to detect translation defects at cellular resolution

  • Implementation: Dissociate zebrafish embryos/tissues, isolate single cells, and perform sequencing

• Live Imaging Techniques:

  • Light sheet microscopy of pelo-fluorescent protein fusions

  • FRAP (Fluorescence Recovery After Photobleaching) to measure pelo dynamics

  • Experimental design: Generate stable transgenic lines with pelo-mEGFP and perform time-lapse imaging during development or under stress conditions

• Translatomics Approaches:

  • Ribosome profiling to identify transcripts affected by pelo deficiency

  • Polysome profiling to detect global translation defects

  • Protocol adaptations: Miniaturized methods for limited tissue samples from zebrafish

These advanced techniques leverage the optical transparency and genetic tractability of zebrafish embryos, enabling unprecedented analysis of pelo function in the context of a developing vertebrate .