SNRPD3 Human, Sf9

What is SNRPD3 and what is its role in cellular processes?

SNRPD3 (Small nuclear ribonucleoprotein D3 polypeptide 18kDa, also known as Sm-D3 or snRNP core protein D3) is a core component of the spliceosome in eukaryotes. It plays an essential role in pre-mRNA splicing and small nuclear ribonucleoprotein biogenesis. SNRPD3 contributes to the recognition of splice sites and catalytic steps of splicing reactions. Recent research has revealed its involvement beyond basic splicing functions, showing its participation in cancer progression pathways, particularly in neuroblastoma where it cooperates with the MYCN oncogene to maintain alternative splicing balance. Alternative splicing occurs at the SNRPD3 locus itself, with two transcript variants encoding the same protein having been identified .

Why is SNRPD3 commonly expressed in Sf9 insect cells for research applications?

Sf9 insect cells provide several significant advantages for recombinant SNRPD3 production. First, this expression system efficiently performs post-translational modifications required for proper protein function, including glycosylation, which is evident in SNRPD3 Human recombinant produced in these cells. Second, the baculovirus expression system used with Sf9 cells typically provides high expression levels of recombinant proteins, making it suitable for producing sufficient quantities for research applications. Third, insect cells offer a eukaryotic environment that facilitates proper protein folding, crucial for maintaining the functional structure of complex proteins like SNRPD3. Finally, the addition of tags such as the 6x His tag at the N-terminus allows for efficient purification using chromatographic techniques, resulting in preparations with greater than 80% purity as determined by SDS-PAGE .

What are the structural characteristics of recombinant SNRPD3 Human produced in Sf9 cells?

Recombinant SNRPD3 Human produced in Sf9 insect cells possesses several distinct structural characteristics. The polypeptide chain has a calculated molecular mass of 14,739 Dalton and undergoes glycosylation in Sf9 cells, contributing to its functional properties. It is typically expressed with a 6x His tag at the N-terminus to facilitate purification and detection. The purified protein appears as a sterile filtered clear solution and is commonly supplied in 20mM HEPES buffer at pH 7.6, with 250mM NaCl and 20% glycerol to maintain stability. The protein contains regions that can bind IgG-type human auto-antibodies, making it useful for immunological studies. When assessed by SDS-PAGE, commercially available preparations typically show greater than 80% purity .

How does SNRPD3 contribute to pre-mRNA splicing mechanisms?

SNRPD3 contributes to pre-mRNA splicing through several critical mechanisms. As a core component of small nuclear ribonucleoproteins (snRNPs), it associates with specific small nuclear RNAs and other Sm proteins to form the structural foundation of snRNPs. Once assembled, SNRPD3 participates in the stepwise assembly of the spliceosome on pre-mRNA targets, helping recognize specific sequences at intron-exon boundaries. Although not directly catalytic itself, SNRPD3 is essential for maintaining the structural integrity of the spliceosome during the transesterification reactions that remove introns and join exons. Recent research indicates that SNRPD3 plays a role in maintaining the fidelity of alternative splicing events, particularly in MYCN-driven contexts such as neuroblastoma. Its depletion has been shown to increase differential splicing, especially affecting cell cycle regulators like BIRC5 and CDK10 .

What experimental applications is recombinant SNRPD3 Human, Sf9 suitable for?

Recombinant SNRPD3 Human, Sf9 is suitable for multiple experimental applications in research settings. In Western blot applications, it can be detected using anti-Sm positive patient sera, making it valuable for autoimmune disease research. For ELISA applications, it can be used at a coating concentration of 0.4-0.8 μg/ml, depending on the specific plate and buffer conditions. The protein is also suitable for biotinylation and iodination, which can enhance detection sensitivity in various immunological assays. In immunological function studies, SNRPD3 binds IgG-type human auto-antibodies and can be used in standard ELISA tests, including checker-board analysis of positive/negative sera panels and CDC international reference sera. Additionally, it serves as a valuable tool for investigating protein-protein interactions relevant to splicing mechanisms and cancer biology, particularly the interactions with MYCN and PRMT5 that have been implicated in neuroblastoma progression .

How does SNRPD3 interact with MYCN in neuroblastoma progression?

The interaction between SNRPD3 and MYCN represents a critical mechanism in neuroblastoma progression. MYCN directly up-regulates SNRPD3 expression, as demonstrated by chromatin immunoprecipitation experiments showing MYCN binding to the SNRPD3 promoter. This binding was found to be 7-fold higher than negative control regions in SK-N-BE(2)-C cells and 2.5-fold higher in KELLY cells, both MYCN-amplified neuroblastoma cell lines. When MYCN expression is experimentally suppressed using doxycycline-inducible systems in SHEP.tet21n cells, SNRPD3 mRNA and protein levels significantly decrease, confirming the regulatory relationship .

At the protein level, MYCN directly binds to SNRPD3, forming a complex that also includes the protein arginine methyltransferase PRMT5. This tripartite interaction leads to increased methylation of SNRPD3. Functionally, the MYCN-SNRPD3 axis maintains the fidelity of alternative splicing events within a narrow range that supports neuroblastoma cell growth. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increases differential splicing, particularly affecting cell cycle regulators like BIRC5 and CDK10. This interaction presents potential therapeutic targets, including SNRPD3 methylation and the protein-protein interface between MYCN and SNRPD3 .

What is the significance of SNRPD3 expression in neuroblastoma patient outcomes?

The clinical correlation is supported by functional studies showing that repression of SNRPD3 expression leads to loss of colony formation in vitro and reduced tumorigenicity in vivo. This suggests that high SNRPD3 expression is not merely a biomarker but actively contributes to aggressive disease behavior. The strong association between high SNRPD3 expression and poor outcomes, combined with its functional role in maintaining cancer cell viability, positions SNRPD3 as a promising therapeutic target, particularly for patients with high SNRPD3-expressing tumors .

How does depletion of SNRPD3 affect cell viability in different neuroblastoma cell lines?

Depletion of SNRPD3 has differential effects on cell viability depending on MYCN amplification status. In MYCN-amplified cell lines such as SK-N-BE(2)-C and KELLY, siRNA-mediated knockdown of SNRPD3 causes a significant reduction in cell viability as measured by alamar blue assays. Cell proliferation, assessed by BrdU incorporation, is also markedly reduced following SNRPD3 depletion in these cell lines. Clonogenic assays demonstrate almost complete abrogation of colony formation capability after SNRPD3 knockdown .

What are the implications of SNRPD3 methylation in cancer pathways?

SNRPD3 methylation has emerged as a significant post-translational modification with important implications for cancer pathways, particularly in neuroblastoma. The protein arginine methyltransferase PRMT5 has been identified as a key enzyme responsible for SNRPD3 methylation, occurring in a complex that also includes the MYCN oncogene in neuroblastoma cells. Methylation of SNRPD3 appears to modulate its function in splicing regulation, with the modified protein maintaining the appropriate balance of alternative splicing events required for cancer cell growth .

This modification presents therapeutic opportunities, as PRMT5 inhibitors such as JNJ-64619178 have shown promising results in reducing SNRPD3 methylation and decreasing cell viability in neuroblastoma cells with high SNRPD3 and MYCN expression. The methylation status of SNRPD3 might also serve as a biomarker for MYCN-driven cancer progression and potentially predict response to therapies targeting the PRMT5-SNRPD3-MYCN axis. The methylation of SNRPD3 represents a point of convergence between epigenetic regulation, transcriptional control, and post-transcriptional processing .

What evidence supports SNRPD3 as a potential therapeutic target in neuroblastoma?

Multiple lines of evidence support SNRPD3 as a promising therapeutic target in neuroblastoma. In vitro studies demonstrate that depletion of SNRPD3 using siRNA or shRNA approaches leads to significant reductions in cell viability, proliferation, and clonogenic capacity in MYCN-amplified neuroblastoma cell lines. More compelling evidence comes from in vivo xenograft experiments with doxycycline-inducible SNRPD3 shRNA in SK-N-BE(2)-C cells, showing that SNRPD3 suppression completely ablated neuroblastoma tumorigenesis and resulted in 100% survival of the experimental animals .

The therapeutic potential is further supported by the finding that SNRPD3 forms a complex with MYCN and PRMT5, with PRMT5-mediated methylation of SNRPD3 being crucial for maintaining the appropriate balance of alternative splicing events. The PRMT5 inhibitor JNJ-64619178 reduces both cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression, suggesting that targeting this pathway could be clinically beneficial. Additionally, the selective effect of SNRPD3 depletion on MYCN/MYC-expressing cells suggests a potential therapeutic window that might reduce side effects compared to more broadly cytotoxic approaches .

What are the optimal storage and handling conditions for SNRPD3 Human, Sf9 preparations?

Proper storage and handling of SNRPD3 Human, Sf9 preparations are critical for maintaining protein integrity and experimental reproducibility. For short-term storage (2-4 weeks), the protein can be kept at 4°C in its original formulation buffer (typically 20mM HEPES buffer pH-7.6, 250mM NaCl, and 20% glycerol). For longer periods, store frozen at -20°C and divide into small aliquots before freezing to minimize freeze-thaw cycles. It is crucial to avoid multiple freeze-thaw cycles as they can significantly reduce protein activity, causing degradation, aggregation, or loss of functional properties .

When preparing working dilutions, use buffers compatible with the intended application. For ELISA applications, dilute to a coating concentration of 0.4-0.8 μg/ml, depending on the type of ELISA plate and coating buffer used. For Western blot applications with anti-Sm positive patient sera, optimize dilutions empirically. Periodically assess protein integrity by SDS-PAGE, with commercial preparations typically having greater than 80% purity. When receiving new shipments, the protein should arrive as a sterile filtered clear solution and should be immediately transferred to appropriate storage conditions .

How can researchers effectively design siRNA or shRNA experiments targeting SNRPD3?

Designing effective siRNA or shRNA experiments targeting SNRPD3 requires careful consideration of several factors. Start by designing multiple siRNAs or shRNAs targeting different regions of the SNRPD3 mRNA, avoiding regions with strong secondary structure that might impede siRNA binding. Previous successful approaches have used two distinct siRNAs targeting SNRPD3 in neuroblastoma cell lines (SK-N-BE(2)-C and KELLY). Include appropriate negative controls (non-targeting siRNA/shRNA) with similar GC content, but be aware that some "non-targeting" controls have been reported to inadvertently silence SNRPD3 .

Confirm knockdown efficiency at both mRNA level (qRT-PCR) and protein level (Western blot), aiming for at least 70-80% reduction in expression for meaningful functional studies. For delivery methods, consider lipid-based transfection reagents for transient knockdown or doxycycline-inducible shRNA systems for stable knockdown, which have been successfully employed in both in vitro and in vivo studies of SNRPD3. Include multiple functional assays such as cell viability (alamar blue), proliferation (BrdU incorporation), and clonogenic capacity, and consider performing RNA-seq to assess global changes in splicing patterns following SNRPD3 knockdown .

What techniques are available for detecting and quantifying SNRPD3 in experimental settings?

Several techniques are available for detecting and quantifying SNRPD3 in experimental settings. Western blotting using anti-SNRPD3 antibodies or anti-Sm positive patient sera is a primary detection method, with the 6x His tag on recombinant SNRPD3 detectable using anti-His antibodies. Recommended antibody concentrations range from 0.04-0.4 μg/mL for immunoblotting. Immunohistochemistry allows detection of SNRPD3 in tissue sections with recommended antibody dilutions of 1:200-1:500, while immunofluorescence provides subcellular localization information with recommended antibody concentrations of 0.25-2 μg/mL .

ELISA techniques can use SNRPD3 with a coating concentration of 0.4-0.8 μg/ml and are suitable for quantitative analysis of antibody binding. RNA-based methods like qRT-PCR can measure SNRPD3 mRNA expression levels, while RNA-seq analysis can detect changes in SNRPD3 expression as well as its effects on global splicing patterns. Mass spectrometry can detect and quantify SNRPD3 protein levels in complex samples and is particularly useful for studying post-translational modifications, such as methylation by PRMT5. Chromatin Immunoprecipitation (ChIP) has been used to study transcription factor binding to the SNRPD3 promoter, revealing 7-fold higher MYCN binding compared to control regions in certain cell lines .

What are the considerations for using SNRPD3 Human, Sf9 in immunological applications?

When using SNRPD3 Human, Sf9 in immunological applications, several important factors must be considered. The protein binds IgG-type human auto-antibodies, making it valuable for autoimmune disease research, particularly systemic lupus erythematosus (SLE) studies. For ELISA applications, the optimal coating concentration ranges from 0.4-0.8 μg/ml, depending on the type of ELISA plate and coating buffer used. The protein is suitable for standard ELISA tests, including checker-board analysis of positive/negative sera panels, and consider including CDC international reference sera for standardization .

Regarding antibody specificity, commercial antibodies against human SNRPD3 may cross-react with rat and mouse homologs due to high sequence conservation, which can be advantageous for comparative studies across species. The standard formulation (20mM HEPES buffer pH-7.6, 250mM NaCl, and 20% glycerol) may need to be adjusted for specific immunological applications. Also consider that the glycosylation of SNRPD3 produced in Sf9 cells and its methylation status may influence antibody recognition and should be considered when interpreting results .

How can researchers assess the functional relationship between SNRPD3 and alternative splicing?

Assessing the functional relationship between SNRPD3 and alternative splicing requires a multifaceted approach. RNA-sequencing following manipulation of SNRPD3 levels (through siRNA, shRNA, or overexpression) represents the gold standard for evaluating global changes in splicing patterns. Previous studies have revealed that MYCN overexpression leads to a global increase in differential splicing, while surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increases differential splicing, particularly of cell cycle regulators like BIRC5 and CDK10 .

To establish causality, researchers can perform rescue experiments with wildtype versus mutant SNRPD3 (such as methylation-deficient variants) in SNRPD3-depleted cells. Protein-protein interaction studies, including co-immunoprecipitation and proximity ligation assays, can assess the interaction between SNRPD3, MYCN, and PRMT5 and how these interactions influence splicing outcomes. Treatment with PRMT5 inhibitors like JNJ-64619178 can reveal the importance of SNRPD3 methylation in splicing regulation. Correlating splicing changes with functional outcomes such as cell viability, proliferation, and in vivo tumorigenesis helps establish the biological significance of SNRPD3-mediated splicing regulation in cancer progression .