Recombinant Saimiri boliviensis boliviensis Ribonuclease-like protein 9 (RNASE9)

Basic Research Questions

• What is Saimiri boliviensis boliviensis RNASE9 and how is it classified taxonomically?

RNASE9 (Ribonuclease-like protein 9) from Saimiri boliviensis boliviensis (Bolivian squirrel monkey) is a member of the ribonuclease superfamily. Taxonomically, Saimiri boliviensis boliviensis is classified as a New World primate . The ribonuclease superfamily consists of both canonical (RNases 1-8) and non-canonical (RNases 9-13) members. RNASE9 belongs to the non-canonical subgroup, which typically lacks ribonucleolytic activity due to insertions, deletions, or mutations affecting active site residues .

Methodology for classification:

• Sequence alignment with other ribonucleases to identify conserved domains

• Phylogenetic analysis to determine evolutionary relationships

• Analysis of structural characteristics including conserved cysteine residues that form disulfide bonds

• Examination of catalytic residues to assess potential enzymatic activity

• What expression systems are typically used to produce recombinant RNASE9?

Recombinant RNASE9 from Saimiri boliviensis boliviensis is typically produced in E. coli expression systems, as evidenced by commercially available preparations . For optimal expression:

• Use prokaryotic expression vectors such as pcDNA3.1+/C-(K)DYK or customized vectors containing appropriate promoters and selection markers

• Transform expression vectors into competent E. coli strains optimized for recombinant protein production

• Induce protein expression under controlled conditions (temperature, IPTG concentration)

• Purify using affinity chromatography, leveraging tags such as C-terminal DYKDDDDK (FLAG) tags

• Validate protein identity through western blotting, mass spectrometry, or enzymatic assays

E. coli-based systems are preferred due to their high yield, cost-effectiveness, and rapid growth characteristics, though proper folding of disulfide bonds in ribonucleases may require specialized strains or post-expression treatment .

• How does RNASE9 differ structurally from canonical ribonucleases?

While canonical ribonucleases (RNases 1-8) possess catalytic activity dependent on key histidine and lysine residues, non-canonical RNases like RNASE9 exhibit several structural differences:

• RNASE9 maintains the basic three-dimensional architecture with conserved cysteine residues that form disulfide bonds supporting protein structure

• Critical catalytic residues for ribonucleolytic activity (equivalent to His12, His119, and Lys41 in RNase A) are typically absent or altered in RNASE9

• Sequence identity with canonical RNases is typically in the 15-30% range

• Despite structural similarities, the alterations in active site residues render RNASE9 catalytically inactive for standard RNA degradation

Researchers can assess these structural differences through:

• Sequence alignment with canonical RNases

• Structural prediction using homology modeling

• Analysis of conserved disulfide bond patterns

• Experimental validation using recombinant protein and activity assays

Advanced Research Questions

• What methodological approaches are recommended for studying potential non-canonical functions of RNASE9?

Despite lacking traditional ribonucleolytic activity, RNASE9 may possess important biological functions. To investigate these:

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity ligation assays in relevant cell types

• Gene Expression Analysis:

  • RNA-seq of tissues with high RNASE9 expression compared to knockdown/knockout models

  • qRT-PCR validation of differentially expressed genes

  • Single-cell transcriptomics to identify cell-specific effects

• Functional Assays:

  • Cell migration/invasion assays to test for effects on cellular motility

  • Receptor binding studies using labeled recombinant RNASE9

  • Assessment of membrane localization through fractionation studies

• Evolutionary Analyses:

  • Comparison of selection pressures on RNASE9 across primate lineages

  • Identification of conserved non-catalytic domains that may mediate novel functions

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM to determine three-dimensional structure

  • Surface plasmon resonance to quantify potential ligand interactions

Non-canonical functions may include receptor signaling (similar to hRNase5/ANG, which can function as an EGFR ligand ), or involvement in reproductive biology, as suggested for some mammalian RNases.

• How can researchers effectively validate the expression patterns of RNASE9 in Saimiri boliviensis tissues?

To characterize RNASE9 expression patterns in squirrel monkey tissues:

• Transcriptomic Analysis:

  • RNA-seq of multiple tissue types from Saimiri boliviensis boliviensis

  • qRT-PCR with primers specific to RNASE9 (differentiating from other RNase family members)

  • In situ hybridization to localize transcript expression in tissue sections

• Protein Detection:

  • Develop specific antibodies against RNASE9 (considering cross-reactivity with other RNases)

  • Immunohistochemistry/immunofluorescence on fixed tissues

  • Western blotting with tissue-specific protein extracts

  • ELISA for quantitative assessment in tissue homogenates or biological fluids

• Single-Cell Analysis:

  • Single-cell RNA-seq to identify cell-specific expression patterns

  • Flow cytometry with specific antibodies in dissociated tissues

• Comparative Analysis:

  • Compare expression patterns between Saimiri species (e.g., S. boliviensis vs. S. sciureus) to identify species-specific differences

  • Cross-reference with human RNASE9 expression data to identify conserved patterns

By combining these approaches, researchers can establish comprehensive expression profiles, which may provide insights into potential biological functions based on tissue localization.

• What are the optimal conditions for the functional characterization of recombinant RNASE9?

For comprehensive functional characterization of recombinant RNASE9:

Buffer and Storage Conditions:

• Test pH range (typically 6.0-8.0) for optimal stability

• Evaluate various buffer compositions (phosphate, Tris, HEPES)

• Include stabilizing agents like glycerol (10-20%) for long-term storage

• Store at -80°C for long-term or -20°C with cryoprotectants for medium-term use

Activity Assessment:

• Despite being predicted as non-catalytic, test for residual ribonucleolytic activity using:

  • Zymogram gels with RNA substrates

  • Fluorescence-based assays with labeled RNA substrates

  • High-sensitivity RNA degradation assays with extended incubation times

Binding Studies:

• Surface plasmon resonance (SPR) to identify potential binding partners

• Pull-down assays with tissue lysates followed by mass spectrometry

• Screening against known ribonuclease inhibitor proteins to assess potential interactions

Structural Validation:

• Circular dichroism to confirm proper protein folding

• Size exclusion chromatography to verify monomer/oligomer status

• Dynamic light scattering to assess aggregation state

Cell-Based Assays:

• MTT/XTT assays to evaluate potential cytotoxicity

• Cell binding assays with labeled RNASE9

• Internalization studies using confocal microscopy

These methodological approaches should be adjusted based on initial findings and hypothesis-driven investigations into RNASE9's biological role.

• How might RNASE9 interact with ribonuclease inhibitor proteins in Saimiri boliviensis?

The interaction between ribonucleases and ribonuclease inhibitor (RI) proteins is crucial for regulating their activity in vivo. For RNASE9:

Structural Considerations:

• Despite lacking catalytic activity, RNASE9 may retain structural features that allow RI binding

• The horseshoe-shaped structure of RI, with its concave interior surface of β-strands, forms the binding interface for ribonucleases

• RNASE9 might interact with RI through conserved surface residues even without catalytic activity

Methodological Approaches:

• Binding Assays:

  • Surface plasmon resonance (SPR) to measure binding kinetics between recombinant RNASE9 and RI

  • Isothermal titration calorimetry to determine thermodynamic parameters

  • ELISA-based binding assays for high-throughput screening

• Structural Studies:

  • X-ray crystallography of RNASE9-RI complex (comparable to the approach used for hRI·RNase 1)

  • Hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

  • Computational modeling based on known RI-ribonuclease complexes

• Mutagenesis Studies:

  • Site-directed mutagenesis of potential RI-binding residues in RNASE9

  • Reciprocal mutations in RI to test specificity

  • Analysis of binding affinity changes through SPR or other quantitative methods

Research on RNase A has shown that RI achieves high affinity for diverse ribonucleases by burying substantial complex surface area (2800-3677 Ų) and using long-range electrostatics . Similar mechanisms might apply to RNASE9 despite its non-canonical nature.

• What evolutionary insights can be gained from studying RNASE9 across primate species?

Evolutionary analysis of RNASE9 across primates offers valuable insights into its biological significance:

Methodological Approaches:

• Sequence Analysis:

  • Multi-species alignment of RNASE9 sequences from diverse primates

  • Calculation of dN/dS ratios to detect selection pressures

  • Identification of conserved domains versus rapidly evolving regions

• Phylogenetic Analysis:

  • Construction of phylogenetic trees to compare RNASE9 evolution with species divergence

  • Comparison with other RNase family members to identify lineage-specific expansions

  • Analysis of gene duplication events in the primate RNase locus

• Structural Conservation:

  • Homology modeling of RNASE9 from different species

  • Mapping of conserved residues onto three-dimensional structures

  • Comparison of predicted surface properties across species

Potential Insights:

• Evolutionary rates may reveal functional constraints or adaptive evolution

• Comparison between Saimiri species (S. boliviensis vs. S. sciureus) may reveal recent selective pressures

• Correlation with Alu insertion polymorphisms in Saimiri, which could affect RNASE9 regulation

• Insights into how non-canonical RNases evolved from canonical ancestors with ribonucleolytic activity

Understanding the evolutionary trajectory of RNASE9 may help predict its biological function and importance in primate biology.

• What approaches can address the challenges in studying RNASE9 function in vivo in the Saimiri boliviensis model?

Working with Saimiri boliviensis as a model organism presents unique challenges that require specialized approaches:

Ethical and Practical Considerations:

• Develop non-invasive methods to study RNASE9 function in captive or wild squirrel monkey populations

• Utilize banked tissues or biological samples from existing repositories

• Consider alternative models for initial studies before validation in squirrel monkeys

Methodological Strategies:

• Cell-Based Systems:

  • Develop primary cell cultures from Saimiri boliviensis tissues

  • Create immortalized cell lines using appropriate transformation methods

  • Implement CRISPR-Cas9 gene editing in derived cell lines

• Ex Vivo Approaches:

  • Utilize organ cultures or tissue explants

  • Develop organoid models from stem cells

  • Design perfusion systems for isolated tissue studies

• Molecular Tools:

  • Design species-specific antibodies against Saimiri RNASE9

  • Develop viral vectors for gene delivery optimized for squirrel monkey cells

  • Create reporter systems to monitor RNASE9 expression in real-time

• Comparative Analysis:

  • Parallel studies in more accessible model organisms

  • Comparative genomics approaches using existing sequence data

  • Cross-species validation of findings

• Non-Invasive Monitoring:

  • Analysis of RNASE9 in biological fluids (e.g., milk , saliva, urine)

  • Correlation of polymorphisms with observable phenotypes

  • Remote health monitoring in existing populations

These approaches can help overcome the limitations of working with non-human primates while generating valuable data on RNASE9 function.

• How might RNASE9 function in reproductive biology, and what experimental designs would best address this question?

Based on the expression patterns of other non-canonical RNases, RNASE9 may play a role in reproductive biology:

Background Context:

• Several non-canonical RNases in mammals are expressed in reproductive tissues

• Despite lacking ribonucleolytic activity, these proteins may serve as signaling molecules or have antimicrobial properties

• Understanding RNASE9's role may provide insights into squirrel monkey reproductive biology

Experimental Approaches:

• Expression Profiling:

  • Comprehensive analysis of RNASE9 expression in reproductive tissues (testis, epididymis, ovary, uterus)

  • Hormonal regulation studies to determine if expression varies with reproductive cycle

  • Single-cell RNA-seq to identify specific cell types expressing RNASE9

• Functional Studies:

  • Sperm-binding assays using recombinant RNASE9

  • Zona pellucida interaction studies

  • In vitro fertilization experiments with and without RNASE9 supplementation

• Antimicrobial Testing:

  • Bacterial and fungal growth inhibition assays

  • Mechanism studies (membrane disruption, metabolic inhibition)

  • Testing against reproductive tract-specific microorganisms

• Comparative Analysis:

  • Correlation of RNASE9 polymorphisms with fertility parameters

  • Cross-species comparison of expression patterns and sequence conservation

  • Evolutionary analysis of selection pressures on reproductive versus non-reproductive tissues

• Receptor Identification:

  • Pull-down assays using reproductive tissue lysates

  • Cross-linking studies to identify binding partners

  • Receptor activation assays to test signaling capabilities