Recombinant Danio rerio Prolyl-tRNA synthetase associated domain-containing protein 1 (Prorsd1)

What is Prolyl-tRNA synthetase associated domain-containing protein 1 (Prorsd1) in zebrafish?

Prorsd1 in zebrafish (Danio rerio) is a protein associated with the aminoacyl-tRNA synthetase family, specifically containing domains related to prolyl-tRNA synthetase functionality. It belongs to the same protein family as those found in other vertebrates such as mice and Xenopus tropicalis, as well as more distantly related homologs in plants like Oryza sativa . The protein likely plays a role in the aminoacylation process where proline is attached to its cognate tRNA during protein synthesis.

Similar to the canonical Prolyl-tRNA synthetase (ProRS), which catalyzes the reaction:
$Pro + tRNA^{Pro} + ATP \rightarrow Pro-tRNA^{Pro} + AMP + PP_i$

While the exact structure of zebrafish Prorsd1 has not been experimentally determined according to available data, functional predictions can be made based on homologous proteins and domain conservation.

How does Prorsd1 differ from canonical Prolyl-tRNA synthetase in zebrafish?

Prorsd1 contains domains associated with prolyl-tRNA synthetase but differs from the canonical enzyme in several key aspects:

Methodologically, researchers can distinguish between these proteins through differential expression analysis during zebrafish development stages, as proteomics studies have shown stage-specific protein expression patterns during embryogenesis . Computational structure prediction using protein-threading approaches similar to those employed for other zebrafish proteins can help elucidate structural differences .

What expression pattern does Prorsd1 exhibit during zebrafish development?

While specific Prorsd1 expression data is not directly provided in the search results, research methodologies for determining expression patterns can be adapted from existing zebrafish studies. Based on proteomics studies of zebrafish embryonic development, protein expression typically follows stage-specific patterns :

Researchers can visualize Prorsd1 expression using color-based or fluorescent-based in situ hybridization. The fluorescent method offers higher sensitivity and allows simultaneous detection of multiple probes, although it requires confocal microscopy equipment .

How can I generate zebrafish models with modified Prorsd1 expression?

Creating zebrafish models with modified Prorsd1 expression can be achieved through CRISPR-Cas technology. The methodology involves:

• gRNA Design: Use online databases such as CHOPCHOP (https://chopchop.cbu.uib.no) to identify optimal guide RNA sequences targeting Prorsd1. Select Cas9 or Cas12a/Cpf1 based on precision requirements .

• Component Preparation: For Cas12a/Cpf1, which offers higher precision:

  • Order custom crRNA targeting Prorsd1

  • Purchase Cas12a/Cpf1 protein from commercial suppliers

  • Prepare microinjection mixture containing appropriate concentrations of both components

• Embryo Injection: Inject 1-cell stage zebrafish embryos with the CRISPR components.

• Mutation Detection: Use fragment analysis through high-resolution capillary gel-electrophoresis to identify indels:

  • Design PCR primers flanking the targeted region (ideally spanning exons)

  • Amplify the region using PCR

  • Analyze fragments using a fragment analyzer platform

  • Identify successful mutations by comparing fragment sizes to wild-type controls

• Confirmation: Perform Sanger sequencing of PCR products from positive samples to confirm the exact nature of mutations .

This method can detect indels as small as 2 base pairs, allowing for precise genotyping of newly-generated mutant lines .

What are the optimal conditions for expressing recombinant Danio rerio Prorsd1 in bacterial systems?

While specific optimization data for Prorsd1 expression is not available in the search results, methodological approaches can be adapted from general recombinant protein expression protocols for zebrafish proteins:

• Codon Optimization: Zebrafish genes often require codon optimization for efficient bacterial expression. Use algorithms that account for E. coli codon bias while preserving key regulatory regions.

• Expression Vector Selection: For Prorsd1, consider vectors with:

  • T7 or tac promoter systems for controlled induction

  • Fusion tags (His, GST, MBP) to enhance solubility and facilitate purification

  • Cleavage sites for tag removal if required for functional studies

• Expression Conditions:

  Parameter	Recommended Range	Optimization Method
  Temperature	16-30°C (test 18°C for initial trials)	Test multiple temperatures in small-scale expression
  Induction time	4-16 hours	Monitor expression over time using SDS-PAGE
  IPTG concentration	0.1-1.0 mM	Test concentration gradient
  Media	LB, TB, or 2xYT	Compare protein yield in different media

• Purification Strategy: Design a two-step purification protocol using affinity chromatography followed by size exclusion chromatography to obtain pure protein for functional assays .

• Quality Control: Verify structural integrity using circular dichroism and thermal shift assays similar to those used for validating other tRNA synthetase proteins .

What high-throughput screening methods can be applied to study Prorsd1 function in zebrafish?

Zebrafish embryos offer an excellent platform for high-throughput screening related to Prorsd1 function. The following methodological approaches can be implemented:

• Morpholino-Based Knockdown:

  • Design antisense morpholinos targeting Prorsd1 mRNA

  • Inject into 1-cell stage embryos

  • Assess developmental phenotypes at multiple timepoints (24h, 48h, 72h, 96h, 120h)

  • Score for specific phenotypes related to translation defects

• Small Molecule Screening:

  • Adapt the fish embryo acute toxicity test (FET) protocol

  • Expose embryos to compound libraries in 24-well plates

  • Assess both general toxicity and specific effects on processes related to protein synthesis

  • Implement automated imaging to quantify phenotypic changes

• CRISPR Screen:

  • Create a library of gRNAs targeting different regions of Prorsd1

  • Inject in combination with Cas9 or Cas12a

  • Use fragment analysis to identify mutations

  • Correlate mutation patterns with observed phenotypes

• High-Content Screening (HCS):

  • Generate transgenic zebrafish expressing fluorescent-tagged Prorsd1

  • Perform automated confocal microscopy to track protein localization

  • Analyze data using machine learning algorithms to identify patterns

  • Correlate with developmental outcomes

This approach leverages the zebrafish's advantages including small size, transparency, external fertilization, and rapid development, which collectively facilitate high-throughput analysis .

How can molecular dynamics simulations be used to predict Prorsd1 interactions with potential inhibitors?

Molecular dynamics (MD) simulations provide valuable insights into Prorsd1 structure-function relationships. A comprehensive approach includes:

• Model Generation:

  • Build a 3D structure of zebrafish Prorsd1 using protein-threading approaches

  • Validate the model using structural assessment tools (PROCHECK, Verify3D)

  • Refine the model through energy minimization procedures

• Simulation Setup:

  • Place the protein in a water box with appropriate counterions

  • Apply AMBER or CHARMM force fields

  • Equilibrate the system (minimization, heating, density equilibration)

  • Run production MD for at least 100 ns

• Inhibitor Docking:

  • Perform virtual screening of potential inhibitors against the binding pocket

  • Calculate binding free energies using MM-PBSA or MM-GBSA methods

  • Identify key residues involved in ligand recognition

• Binding Site Analysis:

  • Generate a table of critical residues based on free energy decomposition

  • Compare with experimental mutagenesis data if available

  • Prioritize compounds based on predicted binding affinity and stability

This approach parallels successful strategies used to identify binding residues in zebrafish NOD1-LRR, where His775, Lys777, Asp803, Ser833, and Ile859 were identified as pivotal for ligand interaction .

What is the impact of sex-specific differences on Prorsd1 expression and function in zebrafish?

Sex-specific differences could significantly impact Prorsd1 research in zebrafish. Methodological considerations include:

• Genetic Mapping Considerations:

  • Recombination rates in zebrafish are dramatically suppressed in male meiosis compared to female meiosis, particularly near centromeres

  • When mapping Prorsd1 variants, researchers should consider:

    • Using female meiosis for positional cloning to maximize genetic map to physical distance ratio

    • Using male meiosis when minimizing recombination is beneficial (e.g., maintaining linked alleles)

• Sex-Specific Expression Analysis:

  • Perform RNA-seq and proteomic analysis of Prorsd1 in male versus female zebrafish

  • Isolate tissues from sex-matched individuals at equivalent developmental stages

  • Use qPCR with sex-specific internal controls for validation

• Experimental Design Implications:

  Research Objective	Recommended Approach	Methodological Rationale
  Fine mapping Prorsd1 variants	Female-based mapping panels	Higher recombination rates provide better resolution
  Maintaining Prorsd1 mutations with linked markers	Male-based mapping approaches	Reduced recombination preserves linkage
  Expression studies	Sex-matched cohorts	Controls for sex-specific expression differences

Understanding these sex-specific differences is crucial for experimental design and interpretation of results when studying Prorsd1 in zebrafish .

How does zebrafish Prorsd1 compare structurally and functionally to human prolyl-tRNA synthetase associated proteins?

Comparative analysis between zebrafish and human prolyl-tRNA synthetase associated proteins provides valuable evolutionary and functional insights:

• Structural Comparison Methodology:

  • Generate homology models for both zebrafish and human proteins

  • Perform structural superimposition to identify conserved domains

  • Calculate root mean square deviation (RMSD) to quantify structural divergence

  • Identify species-specific insertions or deletions

• Functional Domain Analysis:

  • Compare domain architecture across species

  • Identify zinc-binding domains and other regulatory regions

  • Assess conservation of catalytic residues

  • Map species-specific variations onto structural models

• Evolutionary Analysis:

  Feature	Human ProRS-Associated Proteins	Zebrafish Prorsd1	Methodological Significance
  Domain Conservation	C-terminal zinc binding domain (~80 aa)	Predicted similar domains	Indicates functional importance across vertebrates
  Catalytic Residues	Conserved ATP-binding motifs	Likely conserved	Suggests preserved biochemical function
  Editing Domains	Present in some isoforms	Variable presence	May indicate species-specific quality control mechanisms

• Translational Relevance:

  • The zebrafish model allows for high-throughput in vivo screening of compounds targeting conserved domains

  • Developmental effects of Prorsd1 disruption can be rapidly assessed

  • Zebrafish embryos provide a vertebrate system with 80% concordance to mammalian developmental toxicity

This comparative approach helps determine whether zebrafish Prorsd1 is an appropriate model for human ortholog studies and identifies potential limitations in translational research .

What are the technical challenges in purifying active recombinant Prorsd1 for biochemical assays?

Purifying active recombinant Prorsd1 presents several technical challenges that researchers must address:

• Solubility Issues:

  • Problem: Prorsd1, like many associated domains of tRNA synthetases, may exhibit poor solubility

  • Solution:

    • Test multiple solubility-enhancing tags (MBP, SUMO, Thioredoxin)

    • Optimize buffer conditions (pH 7.0-8.5, NaCl 150-500 mM)

    • Include stabilizers (5-10% glycerol, 1-5 mM DTT or TCEP)

    • Consider co-expression with molecular chaperones

• Activity Preservation:

  • Problem: Maintaining native conformation and activity during purification

  • Solution:

    • Implement gentle purification conditions (4°C, avoid harsh elution)

    • Include cofactors (ATP, Mg²⁺) in purification buffers

    • Verify structural integrity using circular dichroism and thermal shift assays

    • Perform activity assays immediately after purification

• Protein Yield Optimization:

  Expression System	Typical Yield	Technical Considerations
  E. coli	1-5 mg/L	Inclusion body formation common; requires refolding
  Insect cells	2-10 mg/L	Better folding; higher cost and complexity
  Mammalian cells	0.5-2 mg/L	Best for post-translational modifications; lowest yield

• Activity Assessment:

  • For Prorsd1 domain-containing proteins, standard aminoacylation assays may not be applicable

  • Alternative approaches include:

    • ATP-PPi exchange assays for partial activities

    • Binding assays with potential interaction partners

    • Thermal shift assays to measure ligand-induced stabilization

These technical considerations parallel those encountered with other tRNA synthetase-associated proteins and require careful optimization for each specific construct .

How can zebrafish models be used to investigate the role of Prorsd1 in disease mechanisms?

Zebrafish models offer unique advantages for investigating Prorsd1's role in disease mechanisms:

• Disease Model Generation:

  • Create Prorsd1 knockout or mutant lines using CRISPR-Cas9

  • Generate transgenic lines with fluorescent reporters under Prorsd1 promoter

  • Establish conditional expression systems using Gal4/UAS or Cre/lox technology

• Phenotypic Analysis Methodology:

  • Perform comprehensive developmental assessment (mortality, hatching, morphological defects)

  • Conduct detailed histological examination, including liver histology

  • Implement behavioral testing for neurological phenotypes

  • Use high-resolution imaging to visualize cellular changes

• Disease-Relevant Assays:

  Disease Context	Zebrafish Assay	Methodological Approach
  Neurological disorders	Motor function, brain development	Automated movement tracking, confocal imaging
  Metabolic dysfunction	Energetics, mitochondrial function	Oxygen consumption, ATP production assays
  Developmental disorders	Embryonic phenotyping	Time-lapse imaging, tissue-specific marker analysis
  Inflammatory conditions	Immune response	Neutrophil migration, cytokine expression

• Therapeutic Screening:

  • Test compounds that may restore function in Prorsd1-deficient models

  • Implement medium to high-throughput screening approaches

  • Establish dose-response relationships

  • Validate findings in mammalian systems

This comprehensive approach leverages zebrafish's high concordance with mammalian systems while offering the advantages of rapid development, optical transparency, and amenability to genetic manipulation .

What bioinformatic resources are available for analyzing zebrafish Prorsd1 sequence, structure, and expression data?

Researchers studying zebrafish Prorsd1 can utilize numerous bioinformatic resources:

• Sequence and Orthology Databases:

  • ZFIN's General Fish Database - Contains zebrafish gene information and expression data

  • Ensembl and NCBI HomoloGene - For identifying orthologs and paralogs

  • The Sanger Institute's Zebrafish Mutation Project - For existing mutations

• Structural Analysis Tools:

  • I-TASSER or PHYRE2 - For protein structure prediction

  • PyMOL or UCSF Chimera - For structural visualization and analysis

  • MDWeb or GROMACS - For molecular dynamics simulations

• Expression Analysis Resources:

  • Expression Atlas - For gene expression across tissues and conditions

  • Single Cell Expression Atlas - For cell-type specific expression patterns

  • ZebrafishMine - For integrating multiple data types

• Methodological Integration Framework:

  Data Type	Recommended Tools	Integration Approach
  Genomic	IGV, UCSC Genome Browser	Visualize genomic context and conservation
  Transcriptomic	DESeq2, EdgeR	Identify differential expression patterns
  Proteomic	MaxQuant, Perseus	Quantify protein abundance changes
  Structural	PyMOL, Chimera	Map variants onto protein structures

• Workflow Implementation:

  • Use Galaxy platform for integrated analysis

  • Implement R/Bioconductor packages for reproducible analysis

  • Apply machine learning approaches for pattern identification

  • Develop visualization pipelines using ggplot2 or Cytoscape

These resources enable comprehensive analysis from sequence to structure to function, facilitating insights into Prorsd1 biology .

How can weighted gene coexpression network analysis (WGCNA) be applied to understand Prorsd1 function in zebrafish development?

WGCNA provides a powerful framework for understanding Prorsd1's functional context within developmental networks:

• Methodological Framework:

  • Generate expression data across multiple developmental timepoints (10+ stages recommended)

  • Perform quality control and normalization of expression data

  • Construct a signed correlation network using appropriate power threshold

  • Identify modules of co-expressed genes

  • Correlate modules with developmental stages and phenotypes

• Prorsd1-Specific Analysis:

  • Determine which module contains Prorsd1

  • Identify hub genes within that module

  • Perform Gene Ontology enrichment analysis of the module

  • Map module genes onto known developmental pathways

• Developmental Stage Correlation:

  Developmental Stage	Typical Module Functions	Analysis Approach
  Early cleavage (4-cell)	Maternal factors, primordial germ cells	Module preservation analysis
  Blastula	Mitochondrial function, energy metabolism	Pathway enrichment analysis
  Gastrulation	Cell migration, pattern formation	Key driver analysis
  Organogenesis	Tissue-specific differentiation	Cell-type enrichment analysis
  5 dpf	Organ function, eye development	Phenotype correlation

• Validation Approaches:

  • Experimental validation of key network predictions

  • Perturbation analysis (knockdown/knockout of Prorsd1)

  • Comparison with human developmental networks

  • Integration with proteomics data

This approach has successfully categorized zebrafish developmental proteins into 11 modules with distinct characteristics and functions during embryogenesis, providing a framework for understanding Prorsd1's role in this process .

What considerations should guide experimental design when comparing wild-type and Prorsd1-mutant zebrafish?

Rigorous experimental design is crucial when comparing wild-type and Prorsd1-mutant zebrafish:

• Genetic Background Considerations:

  • Use siblings from the same crosses whenever possible

  • Backcross mutant lines to wild-type for at least 3-5 generations

  • Consider the strain background (AB, TU, or WIK) and its specific characteristics

  • Account for potential off-target effects of CRISPR-Cas9

• Statistical Power Analysis:

  • Perform a priori power calculations to determine sample size

  • For developmental phenotyping, minimum 30-50 embryos per group

  • For molecular analyses, minimum 3-5 biological replicates

  • For behavioral studies, minimum 12-16 animals per group

• Controls and Validation:

  Control Type	Purpose	Implementation
  Wild-type siblings	Direct genetic background control	Use siblings from heterozygous crosses
  Rescue experiments	Validate specificity of phenotype	Inject Prorsd1 mRNA into mutant embryos
  Alternative mutant alleles	Confirm phenotype reproducibility	Generate multiple mutant lines
  Pharmacological validation	Complement genetic approach	Use inhibitors of related pathways

• Phenotypic Analysis Depth:

  • Assess across multiple developmental timepoints (24h, 48h, 72h, 96h, 120h)

  • Employ multiple methodologies (morphology, histology, behavior, molecular)

  • Use quantitative metrics whenever possible

  • Implement blinded scoring to prevent bias

• Molecular Analysis Framework:

  • Perform comprehensive transcriptomics (RNA-seq)

  • Compare proteome changes using label-free quantitative proteomics

  • Analyze metabolic changes that may result from translation defects

  • Integrate multiple omics datasets