Recombinant Crassostrea gigas Ribonuclease Oy

What are the key ribonucleases identified in Crassostrea gigas?

Several important ribonucleases have been identified in C. gigas, with CgDicer being one of the most well-characterized. CgDicer functions as a member of the ribonuclease III family and plays a critical role in the RNA interference (RNAi) pathway. Additionally, the RIG-I-like receptor (RLR) homolog, designated as CgRIG-I, has been identified as an important component of antiviral immunity. These ribonucleases are instrumental in sequence-specific degradation of RNA molecules and participate in defense against viral infections. CgDicer contains two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM), while CgRIG-I contains caspase activation and recruitment domains (CARDs), an RNA helicase domain, and a C-terminal regulatory domain (RD) .

What is the structure and function of CgDicer in Crassostrea gigas?

CgDicer is a substantial protein with a polypeptide length of 1873 amino acids. Its molecular structure includes two critical functional domains: the double-stranded RNA-binding motif (DSRM) and ribonuclease III domains (RIBOc). Functionally, CgDicer serves dual roles in the oyster's cellular machinery. First, it operates as an intracellular recognition molecule that can bind to double-stranded RNA (dsRNA). Second, it functions as an effector with ribonuclease activity, capable of cleaving dsRNA molecules. These activities are central to the RNA interference pathway, where CgDicer directs the sequence-specific degradation of complementary mRNA. The enzyme shares sequence identities ranging from 18.5% to 46.6% with Dicer proteins identified in other organisms, indicating evolutionary conservation of this critical function while maintaining species-specific adaptations .

How is CgRIG-I structured and what is its significance in C. gigas immunity?

The CgRIG-I gene produces a full-length cDNA of 3436 bp, including 5'- and 3'-untranslated regions (UTRs) of 93 bp and 286 bp respectively, with an open reading frame (ORF) of 3057 bp. This gene encodes a substantial polypeptide of 1018 amino acids with an estimated molecular mass of 116.5 kDa. SMART analysis has revealed that the CgRIG-I protein contains the conserved domains typically found in RIG-I proteins, including caspase activation and recruitment domains (CARDs), an RNA helicase domain, and a C-terminal regulatory domain (RD). Phylogenetic analysis positions CgRIG-I within the clade of its vertebrate homologs, suggesting evolutionary conservation of this important immune receptor. CgRIG-I is pivotal for detecting viral nucleic acids and mediating innate immune responses, demonstrating responsiveness to poly(I:C) stimulation but not to other pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides .

How does CgDicer expression respond to immunological stimuli?

CgDicer expression demonstrates significant responsiveness to immunological challenges, particularly those involving double-stranded RNA. In experimental studies, the mRNA expression level of CgDicer in hemocytes was dramatically upregulated by 36.70 ± 11.10 fold (p < 0.01) following treatment with double-stranded RNA (dsRNA). This substantial increase indicates the enzyme's critical role in dsRNA processing and antiviral response mechanisms. In primary cultures of oyster hemocytes, CgDicer transcripts were significantly induced 12 hours after poly(I:C) stimulation, reaching levels 2.04-fold higher (p < 0.05) than those in control groups. This time-dependent induction profile suggests a regulated response mechanism that activates following detection of viral-like molecular patterns. The differential expression across various challenge conditions highlights CgDicer's specific role in antiviral immunity rather than general immune responsiveness .

What experimental evidence supports the dual function of CgDicer in dsRNA binding and cleavage?

The multifunctional nature of CgDicer has been experimentally validated through the recombinant expression and functional characterization of its key domains. The DSRM (double-stranded RNA-binding motif) and RIBOc (ribonuclease III domain) were separately expressed in Escherichia coli transetta (DE3) to evaluate their specific functions. The recombinant DSRM protein demonstrated significant binding activity to both dsRNA and poly(I:C) in vitro, confirming its role as a pattern recognition receptor for double-stranded RNA molecules. Complementarily, the recombinant RIBOc protein exhibited significant dsRNase activity, effectively cleaving dsRNA substrates in vitro. These experimental findings provide compelling evidence for CgDicer's dual functionality: first recognizing and binding to dsRNA through its DSRM domain, then executing catalytic cleavage via its RIBOc domain. This bifunctional capability enables CgDicer to serve as both a sensor and effector in antiviral defense mechanisms, making it a versatile component of the oyster's innate immune system .

How do transcriptomic and metabolomic approaches inform our understanding of C. gigas defense mechanisms?

Integrated transcriptomic and metabolomic analyses have revealed complex molecular mechanisms underlying C. gigas immunity and growth-defense trade-offs. Principal component analysis of transcriptomic data has shown distinct separation between C. gigas and related species, with 2,006 differentially expressed genes (DEGs) identified. Gene Ontology enrichment analysis of these DEGs has highlighted biological processes related to immune function, cellular components, and molecular functions relevant to defense responses. Similarly, metabolomic analysis using LC-QTOF-MS identified 3,149 metabolites (2,464 in positive and 685 in negative ionization modes), with orthogonal projections to latent structures–discriminate analysis (OPLS-DA) models showing robust parameters (R2X = 0.492 and 0.637, R2Y = 0.965 and 0.961, and Q2 = 0.784 and 0.86 for positive and negative ionization modes). These multi-omics approaches have demonstrated how C. gigas allocates energy resources between growth and defense functions, providing a comprehensive view of the molecular basis for immune response in this economically important mollusk .

What techniques are most effective for evaluating ribonuclease activity in C. gigas?

Multiple complementary techniques have proven effective for evaluating ribonuclease activity in C. gigas, each providing unique insights into enzyme function and expression. RT-qPCR using gene-specific primers represents a primary approach for quantifying ribonuclease transcript levels across different tissues or experimental conditions. For functional characterization, recombinant expression of specific domains (such as DSRM and RIBOc) in bacterial systems like E. coli transetta (DE3) allows for the isolation and testing of distinct enzymatic activities. In vitro binding and cleavage assays using purified recombinant proteins and RNA substrates provide direct evidence of ribonuclease function. Immunocytochemistry offers valuable information on subcellular localization, which informs understanding of the enzyme's biological context. For comprehensive analysis, integrated approaches combining transcriptomics (to identify expression patterns) and metabolomics (to assess downstream effects) provide holistic insights into ribonuclease activity within broader biological processes .

How can researchers effectively preserve RNA integrity when working with ribonuclease samples?

The preservation of RNA integrity is critical when studying ribonucleases, as sample degradation can significantly impact experimental outcomes. Based on research with ribonuclease inhibitors, effective strategies include the addition of RNase inhibitors to RNA samples immediately following purification. In experimental designs where ribonuclease activity needs to be preserved for later analysis, storage at –20°C with ribonuclease inhibitor supplementation has been shown to maintain RNA integrity even after 6 weeks of storage with multiple freeze-thaw cycles. Quantitative assessments of RNA quality should employ multiple methodologies, including spectrophotometry (using A260/A230 and A260/A280 ratios to assess purity), fluorescence-based quantitation systems, and capillary electrophoresis (such as Agilent Bioanalyzer) to verify RNA integrity. For downstream applications like RT-qPCR, preliminary testing should confirm that protection strategies do not interfere with amplification efficiency. This approach ensures reliable results across experimental timelines and facilitates reproducible research with ribonuclease samples .

What controls should be implemented when studying C. gigas ribonucleases?

When studying C. gigas ribonucleases, implementing robust controls is essential for result validation and interpretation. For gene expression studies, researchers should include multiple reference genes that maintain stable expression across experimental conditions, tissues, and developmental stages. When evaluating ribonuclease activity, both positive controls (known ribonuclease substrates) and negative controls (heat-inactivated enzymes or samples without enzymes) should be incorporated to establish baseline activity levels and confirm specific enzymatic action. For binding assays, controls should include non-specific RNA or DNA sequences to demonstrate binding specificity. Time-course experiments are valuable for distinguishing between immediate and delayed responses to stimuli, requiring untreated controls at each time point. When using recombinant proteins, parallel experiments with purified buffer components help distinguish between effects of the active protein and those of buffer constituents or contaminants. For in vivo challenges, mock-treated controls and dose-response relationships should be established to confirm specific effects of the tested compounds or conditions .

Table 1: Expression changes of CgDicer in response to immune stimulation

Table 2: Functional domains of key ribonucleases in C. gigas

Table 3: Comparison of techniques for RNA integrity assessment