Recombinant Bovine Prolyl-tRNA synthetase associated domain-containing protein 1 (PRORSD1)

What is PRORSD1 and what functional domains characterize this protein?

PRORSD1 (Prolyl-tRNA synthetase domain containing 1) is a protein characterized by the presence of a YbaK domain, which is typically associated with tRNA editing activity . While much of the current research focuses on murine models, the bovine ortholog shares significant structural homology. The protein contains domains responsible for aminoacyl-tRNA editing activity, which plays a crucial role in translational fidelity by hydrolytically removing incorrectly attached amino acids from tRNAs .

The functional architecture of PRORSD1 includes:

• YbaK domain for tRNA editing functionality

• Structural motifs for protein-protein interactions

• Motifs associated with molecular recognition mechanisms

Research indicates that PRORSD1 functions within a network of proteins involved in tRNA processing and quality control during protein synthesis, suggesting its importance in maintaining translational accuracy.

What methodological approaches are most effective for recombinant expression of bovine PRORSD1?

Based on expression systems used for murine PRORSD1, researchers have found several effective approaches for recombinant protein production:

For optimal expression of bovine PRORSD1:

• Consider using mammalian expression systems such as HEK293 cells, which have been successful with mouse PRORSD1

• Incorporate affinity tags (His, Fc, or Avi) to facilitate purification while maintaining protein function

• Optimize codon usage for the expression system selected

• Include protease inhibitors during purification to prevent degradation

The choice between bacterial and mammalian expression systems should be guided by the specific experimental requirements, particularly whether post-translational modifications are critical for the intended application.

How can researchers effectively assess the aminoacyl-tRNA editing activity of PRORSD1?

To evaluate the aminoacyl-tRNA editing activity of PRORSD1, researchers can employ several complementary approaches:

• In vitro deacylation assays: Using radiolabeled or fluorescently labeled misacylated tRNAs as substrates to measure the rate of hydrolysis catalyzed by purified PRORSD1.

• Coupled enzyme assays: Monitoring AMP production during deacylation reactions using auxiliary enzymes like adenylate kinase and pyruvate kinase/lactate dehydrogenase to convert changes in AMP concentration to measurable NADH oxidation.

• Mass spectrometry-based assays: Quantifying the relative abundance of correctly charged versus misacylated tRNAs before and after incubation with PRORSD1.

When designing these experiments, it's important to include appropriate controls:

• Heat-inactivated PRORSD1 (negative control)

• Known tRNA editing enzymes like DTD1 or DTD2 (positive control)

• Variants of PRORSD1 with mutations in the YbaK domain

These methodological approaches have been applied to proteins with similar functions, including VARS2, AARS, and DTD1, which are documented to share aminoacyl-tRNA editing activity with PRORSD1 .

What approaches are recommended for identifying proteins that interact with PRORSD1?

For characterizing the PRORSD1 interactome, researchers should consider employing multiple complementary techniques:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged PRORSD1 (His, Fc, or Avi tags have been successfully used )

  • Perform pull-down experiments under physiologically relevant conditions

  • Identify interacting partners by mass spectrometry

  • Validate using reciprocal pull-downs with identified partners

• Proximity-based labeling approaches:

  • BioID or TurboID fusion with PRORSD1 to biotinylate nearby proteins

  • APEX2 fusion for proximity-based labeling in specific cellular compartments

  • These methods are particularly valuable for capturing transient interactions

• Yeast two-hybrid screening:

  • Useful for initial discovery of potential interacting partners

  • Results should be validated using co-immunoprecipitation or pull-down assays

• Co-immunoprecipitation from native tissues:

  • Using specific antibodies against endogenous PRORSD1

  • Can identify physiologically relevant interactions in the appropriate tissue context

These methods have been successfully applied to identify protein interactions for related proteins within the tRNA processing machinery , and similar approaches would be applicable to bovine PRORSD1.

How is PRORSD1 potentially involved in neuronal function and what experimental designs would best explore this relationship?

While the exact role of PRORSD1 in neuronal function remains to be fully elucidated, several lines of evidence suggest potential involvement:

• Expression data indicates presence in neuronal tissues, including dorsal root and trigeminal ganglion nociceptors

• Its tRNA editing function would be critical in neurons, which are particularly sensitive to proteostasis disruptions

• Systems genetic analysis has implicated genes with similar functions in hippocampal neuroanatomy and spatial learning

To investigate PRORSD1's role in neuronal function, researchers could employ:

Cellular approaches:

• Primary neuronal cultures with PRORSD1 knockdown or overexpression

• Analysis of neurite growth, synapse formation, and electrophysiological properties

• Compartmentalized chambers to study PRORSD1 function in different neuronal regions

Animal model approaches:

• Conditional knockout models targeting PRORSD1 in specific neuronal populations

• Behavioral assays to assess cognitive and sensory functions

• Analysis of neuronal morphology and connectivity in knockout versus control animals

Molecular approaches:

• Ribosome profiling to assess translation fidelity in neurons with altered PRORSD1 levels

• Proteomics to identify changes in the neuronal proteome when PRORSD1 function is perturbed

• Single-cell transcriptomics to identify cell-type specific effects

These approaches could help elucidate whether PRORSD1 has specific roles in neuronal development, function, or maintenance beyond its general involvement in translation quality control.

What is known about the evolutionary conservation of PRORSD1 and how can this inform functional studies?

The evolutionary conservation of PRORSD1 provides important insights into its fundamental biological functions:

• The YbaK domain found in PRORSD1 is evolutionarily ancient, with homologs present across prokaryotes and eukaryotes, suggesting a fundamental role in translation

• Comparative genomic analysis indicates that PRORSD1 (MGI:1915189) is located on chromosome 11 in mice , with orthologs present across mammalian species

• The conservation of function in aminoacyl-tRNA editing activity across species suggests strong evolutionary pressure to maintain translational fidelity

To leverage evolutionary conservation in functional studies:

• Comparative biochemical analysis:

  • Purify PRORSD1 orthologs from evolutionarily divergent species

  • Compare substrate specificity and catalytic efficiency

  • Identify conserved versus species-specific functions

• Domain swapping experiments:

  • Create chimeric proteins with domains from different species

  • Assess which domains confer species-specific functions

  • Identify the minimal functional unit conserved across evolution

• Rescue experiments in model organisms:

  • Test whether bovine PRORSD1 can functionally replace orthologs in other species

  • Determine whether function depends on species-specific interacting partners

This evolutionary perspective can help distinguish the core conserved functions of PRORSD1 from species-specific adaptations, facilitating more targeted experimental design.

How should researchers analyze differential expression of PRORSD1 in RNA-seq datasets?

When analyzing PRORSD1 expression in RNA-seq data, several methodological considerations are important:

• Proper normalization and statistical testing:

  • Standard t-tests assuming unequal variances between experimental groups have been used in previous studies examining PRORSD1 expression

  • Apply appropriate multiple testing corrections when examining expression across conditions

  • Consider using methods like DESeq2 or EdgeR specifically designed for RNA-seq count data

• Isoform-level analysis:

  • Be aware that PRORSD1 has several synonyms in databases (Prdxdd1, Ncrna00117, RP23-92B18.3, 2010316F05Rik)

  • Examine splice variants and alternative UTR usage that might affect function

  • Consider 3'UTR length variations which have been shown to affect miRNA binding sites in similar genes

• Co-expression network analysis:

  • Identify genes whose expression patterns correlate with PRORSD1

  • Examine co-expressed genes for functional enrichment using tools like GSEA

  • Consider weighted gene correlation network analysis (WGCNA) to identify modules of co-regulated genes

• Integration with other data types:

  • Correlate expression with phenotypic traits in systems genetics approaches

  • Integrate with proteomic data to determine if transcript and protein levels correlate

  • Combine with ChIP-seq data to identify potential transcriptional regulators

Following the methodologies employed in systems genetic analyses , researchers should consider both fold change (with thresholds typically set at >1.5) and statistical significance (q-value < 0.05) when identifying differential expression of PRORSD1 across conditions.

What analytical approaches are appropriate for evaluating PRORSD1's role in complex biological pathways?

To elucidate PRORSD1's role in biological pathways, several complementary analytical approaches are recommended:

• Pathway enrichment analysis:

  • When analyzing proteins that interact with PRORSD1, pathway analysis can reveal biological processes they participate in

  • Tools like KEGG, Reactome, or Gene Ontology can identify overrepresented pathways

  • This approach has identified molecular function and aminoacyl-tRNA editing activity as key functional categories for PRORSD1

• Network analysis:

  • Construct protein-protein interaction networks centered on PRORSD1

  • Identify hub proteins and network modules that might be affected by PRORSD1 perturbation

  • Apply centrality measures to quantify PRORSD1's importance within these networks

• Quantitative trait locus (QTL) mapping:

  • Systems genetic approaches like those used in mouse studies can link PRORSD1 genetic variation to phenotypic traits

  • Distinguish between expression QTLs (eQTLs) affecting PRORSD1 levels and QTLs where PRORSD1 mediates effects on downstream phenotypes

• Multi-omics integration:

  • Integrate transcriptomic, proteomic, and metabolomic data to build comprehensive models

  • Apply methods like partial least squares path modeling or Bayesian networks to infer causal relationships

  • Use these integrated models to generate testable hypotheses about PRORSD1 function

These analytical approaches should be applied in a hypothesis-driven manner, with careful consideration of appropriate controls and statistical power to detect biologically meaningful effects.

How might CRISPR-Cas9 techniques be optimized for studying PRORSD1 function?

CRISPR-Cas9 technology offers powerful approaches for interrogating PRORSD1 function through precise genetic manipulation:

• Generation of knockout cell lines:

  • Design guide RNAs targeting exons encoding the YbaK domain

  • Validate knockout efficiency at both mRNA and protein levels

  • Confirm phenotypes with rescue experiments expressing wildtype PRORSD1

  • Based on successful CRISPR-Cas9 applications in other studies , consider targeting multiple exons simultaneously

• Domain-specific mutations:

  • Create specific mutations in the YbaK domain to dissect function

  • Use homology-directed repair to introduce point mutations that affect activity but not structure

  • Generate truncation mutants similar to those described in related studies to identify minimal functional domains

• Tagged endogenous PRORSD1:

  • Introduce fluorescent or affinity tags at the endogenous locus

  • Preserve native expression patterns and regulation

  • Enable live-cell imaging and purification of native complexes

• Inducible systems:

  • Employ CRISPRi/CRISPRa to modulate PRORSD1 expression

  • Use inducible promoters to control timing of expression changes

  • Apply tissue-specific Cas9 expression for in vivo studies

When designing CRISPR experiments, researchers should consider potential off-target effects and include appropriate controls. The efficacy of this approach has been demonstrated in studies targeting transcription factors with similar experimental complexity .

What emerging technologies might advance our understanding of PRORSD1 function in translation quality control?

Several cutting-edge technologies hold promise for elucidating PRORSD1's precise role in translation:

• Ribosome profiling with misincorporation detection:

  • Adapt ribosome profiling to detect translation errors

  • Compare error rates in cells with normal versus altered PRORSD1 levels

  • Identify specific codons or sequence contexts prone to errors when PRORSD1 function is compromised

• Cryo-electron microscopy:

  • Determine the structure of PRORSD1 alone and in complex with tRNAs

  • Visualize conformational changes during the editing reaction

  • Identify structural features that determine substrate specificity

• Single-molecule techniques:

  • Apply single-molecule fluorescence to monitor PRORSD1-tRNA interactions in real-time

  • Use optical tweezers or AFM to measure binding forces and kinetics

  • Characterize the dynamics of PRORSD1 association with ribosomes or other translation factors

• Spatial transcriptomics and proteomics:

  • Map the subcellular localization of PRORSD1 and its associated proteins

  • Determine whether PRORSD1 function is compartmentalized within cells

  • Identify cell types with particularly high PRORSD1 expression or activity

• Metabolic labeling approaches:

  • Use bioorthogonal amino acid analogs to measure translation fidelity

  • Apply SILAC or TMT labeling to quantify changes in the proteome upon PRORSD1 perturbation

  • Employ puromycin-associated nascent chain proteomics to identify nascent polypeptides affected by PRORSD1

These technologies, while technically challenging, offer unprecedented resolution for understanding PRORSD1's molecular function and could reveal unexpected roles beyond its annotated aminoacyl-tRNA editing activity .