SNRPC Human, Sf9

What is SNRPC and what is its primary biological function?

SNRPC is a member of the U1 small nuclear ribonucleoprotein C family, functioning as a critical component of the U1 small nuclear ribonucleoprotein (snRNP) particle required for spliceosome formation . The protein participates in nuclear precursor messenger RNA splicing, a fundamental step in eukaryotic gene expression . SNRPC, also known as U1-C or U1C, plays a specific role in recognizing and binding to the 5' splice site of pre-mRNA, which initiates the splicing process . This protein is particularly significant in research contexts because snRNP particles are frequently targeted by autoantibodies produced by patients with connective tissue diseases .

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

SNRPC Human Recombinant produced in Sf9 is a glycosylated polypeptide chain with a calculated molecular mass of approximately 25,000 Dalton . The protein is expressed with a 6x His tag at the N-terminus, facilitating purification and detection in experimental settings . The purified protein typically appears as a sterile filtered clear solution and demonstrates greater than 90% purity as determined by SDS-PAGE analysis . The glycosylation pattern in Sf9-expressed SNRPC differs from mammalian expression systems, which can impact certain functional studies but provides advantages for structural analyses due to more homogeneous glycosylation.

How should SNRPC Human recombinant protein be stored and handled to maintain optimal activity?

For optimal stability, SNRPC should be stored at 4°C if the entire vial will be used within 2-4 weeks . For longer-term storage, the protein should be kept frozen at -20°C . Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of activity . The formulation of SNRPC in 20mM HEPES buffer (pH-7.5) with 0.01mM EDTA and 0.02% SDS helps maintain stability but researchers should consider adding protein stabilizers like glycerol (10-15%) for prolonged storage . When working with the protein, maintaining a consistent temperature and avoiding excessive mechanical stress (vigorous vortexing) is recommended to preserve functional integrity.

What are the optimal conditions for expressing SNRPC in Sf9 insect cells?

Expressing SNRPC in Sf9 cells typically involves using a baculovirus expression system optimized for pH, temperature, and infection timing. The optimal MOI (multiplicity of infection) for SNRPC expression typically ranges from 2-5, with protein expression peaking at 48-72 hours post-infection . Temperature control is critical, with optimal expression occurring at 27-28°C. For maximal yield, the culture medium should be supplemented with appropriate nutrients and maintained at pH 6.2-6.4 during the infection phase. Oxygen saturation should be maintained above 30% to prevent hypoxic stress that could affect protein folding. Expression with an N-terminal His-tag facilitates subsequent purification steps while minimizing interference with SNRPC's functional domains .

What purification strategies are most effective for SNRPC from Sf9 expression systems?

The most effective purification protocol for His-tagged SNRPC from Sf9 cells involves a multi-step approach:

• Initial cell lysis using a buffer containing 20mM HEPES (pH 7.5), 300mM NaCl, 10mM imidazole, and protease inhibitors

• Nickel-affinity chromatography utilizing the N-terminal His-tag, with a gradient elution of imidazole (20-250mM)

• Size-exclusion chromatography to remove aggregates and achieve >90% purity

• Optional ion-exchange chromatography for research requiring ultra-high purity

This strategy yields SNRPC with >90% purity as confirmed by SDS-PAGE analysis . For studies requiring native conditions, detergent concentration should be minimized, and purification should be performed at 4°C to maintain protein structure and activity. The final preparation should be dialyzed into a storage buffer containing 20mM HEPES (pH-7.5), 0.01mM EDTA, and 0.02% SDS .

How can researchers effectively validate the functionality of purified SNRPC?

Functional validation of purified SNRPC should include:

Additionally, researchers should verify proper folding through limited proteolysis assays and assess oligomerization state using native PAGE or analytical ultracentrifugation. For definitive functional validation, the purified SNRPC should demonstrate the ability to complement splicing activity in SNRPC-depleted nuclear extracts.

How does SNRPC contribute to biomolecular condensation and nuclear body formation?

SNRPC plays a notable role in biomolecular condensation processes that contribute to nuclear body formation, particularly in splicing-related structures such as Cajal bodies . While SNRPC itself may not be a primary driver of condensation, it interacts with proteins like SMN (survival of motor neurons) that possess Tudor domains capable of binding to dimethylated arginine residues . This recognition of post-translational modifications by folded domains represents a novel mechanism for forming the high-avidity networks required for condensation . SNRPC likely participates in these networks through its interaction with other splicing factors, contributing to the formation of functional condensates where splicing components are concentrated . The dynamic assembly and disassembly of these structures are regulated by factors including RNA binding, protein-protein interactions, and post-translational modifications like methylation and phosphorylation .

What role does SNRPC play in the assembly and function of the spliceosome complex?

SNRPC is a crucial component of the U1 snRNP, which initiates spliceosome assembly by recognizing the 5' splice site. The specific functions of SNRPC in spliceosome assembly include:

• Stabilizing the interaction between U1 snRNA and the 5' splice site through its zinc finger domain

• Recruiting additional spliceosomal components through protein-protein interactions

• Facilitating conformational changes necessary for progression through the splicing cycle

• Potentially regulating alternative splicing decisions through differential interactions

Research indicates that SNRPC undergoes dynamic associations and dissociations during the splicing cycle, with its interaction network changing as splicing progresses . Studies of spliceosome assembly kinetics reveal that SNRPC-mediated interactions occur within milliseconds of pre-mRNA binding, highlighting its role in the earliest stages of splice site recognition.

How do post-translational modifications affect SNRPC function in RNA processing?

Post-translational modifications (PTMs) critically regulate SNRPC's activity in RNA processing. Several key modifications have been identified:

These modifications can influence SNRPC's role in splice site selection and spliceosome assembly . For example, arginine methylation can promote interaction with the Tudor domains found in proteins like SMN, facilitating the incorporation of SNRPC into higher-order structures like Cajal bodies . Phosphorylation can alter SNRPC's binding affinity for RNA and protein partners, potentially serving as a regulatory mechanism during different cellular states or in response to signaling pathways. These PTMs represent important control points for modulating splicing activity and specificity.

What advanced imaging techniques are most suitable for studying SNRPC localization and dynamics?

For studying SNRPC localization and dynamics, researchers should employ a combination of advanced imaging techniques:

• Super-resolution microscopy (SRM) techniques:

  • Stimulated Emission Depletion (STED) microscopy provides resolution down to 30-50 nm, suitable for visualizing SNRPC within nuclear bodies

  • Photoactivated Localization Microscopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM) offer single-molecule resolution (10-20 nm) ideal for studying individual SNRPC molecules

• Live-cell imaging approaches:

  • Fluorescence Recovery After Photobleaching (FRAP) to measure SNRPC mobility and residence time in nuclear bodies

  • Fluorescence Correlation Spectroscopy (FCS) for analyzing diffusion properties and molecular interactions

  • Single-particle tracking (SPT) to follow individual SNRPC molecules in real-time

• Correlative light and electron microscopy (CLEM) to combine functional information from fluorescence imaging with ultrastructural details from electron microscopy

These techniques should be paired with appropriate labeling strategies, including site-specific fluorescent protein fusions or smaller tags like HaloTag or SNAP-tag to minimize interference with SNRPC function . For optimal results, researchers should use photoconvertible fluorophores that allow pulse-chase experiments to track newly synthesized versus mature SNRPC pools.

How can researchers effectively study SNRPC interactions with other spliceosomal components?

To comprehensively study SNRPC interactions with other spliceosomal components, researchers should employ a multi-method approach:

• Biochemical methods:

  • Co-immunoprecipitation (Co-IP) coupled with mass spectrometry to identify interaction partners

  • Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling to capture transient interactions

  • Cross-linking and immunoprecipitation (CLIP) techniques to identify RNA binding sites

• Biophysical approaches:

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Structural methods:

  • Cryo-electron microscopy (cryo-EM) for visualizing SNRPC within the spliceosome complex

  • Nuclear magnetic resonance (NMR) for studying dynamic interactions in solution

  • X-ray crystallography for high-resolution structure determination of SNRPC complexes

Researchers should design experiments that capture both stable and transient interactions, including those that occur only during specific stages of the splicing cycle . The use of catalytically inactive or conformationally restricted mutants can help trap specific interaction states for detailed characterization.

What are the most effective approaches for studying SNRPC's role in liquid-liquid phase separation and biomolecular condensation?

To investigate SNRPC's role in liquid-liquid phase separation (LLPS) and biomolecular condensation, researchers should implement a systematic approach:

• In vitro reconstitution experiments:

  • Purified component mixing assays with controlled concentration, salt, and temperature conditions

  • Measurement of partition coefficients to determine SNRPC enrichment in condensates

  • Microrheology studies to characterize the material properties of formed condensates

• Analysis of condensate dynamics:

  • Fluorescence recovery after photobleaching (FRAP) to measure molecular exchange rates

  • Single-molecule tracking to analyze diffusion dynamics within condensates

  • Temperature-dependent formation and dissolution studies to establish phase diagrams

• Molecular determinants investigations:

  • Mutation analysis of potential multivalent interaction sites in SNRPC

  • Effect of post-translational modifications, particularly arginine methylation which can influence Tudor domain interactions

  • RNA dependency tests using RNase treatment or RNA binding-deficient mutants

When studying SNRPC in the context of biomolecular condensation, it's crucial to examine its interactions with known condensate-forming proteins like SMN, which contains Tudor domains capable of recognizing methylated arginine residues . The study of how these interactions contribute to the formation of higher-order structures like Cajal bodies provides insights into both physiological function and potential pathological aggregation in disease states.

How are SNRPC mutations or dysfunctions associated with human diseases?

SNRPC dysfunction has been implicated in several human disease contexts, particularly those involving RNA processing abnormalities. While direct mutations in SNRPC are relatively rare, alterations in its expression, localization, or post-translational modifications can contribute to disease pathologies. Auto-antibodies targeting snRNP components including SNRPC are frequently observed in connective tissue diseases such as systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) . These autoimmune responses can disrupt normal splicing operations and contribute to disease pathogenesis.

In neurodegenerative contexts, SNRPC dysfunction may play a role through its association with proteins like SMN, mutations in which cause spinal muscular atrophy . Disruptions in biomolecular condensation mechanisms involving SNRPC-interacting proteins can lead to aberrant protein aggregation and cellular toxicity . Understanding these disease associations requires careful analysis of splicing patterns in patient samples and correlation with SNRPC abundance, localization, and modification state.

What experimental models are most appropriate for studying SNRPC function in disease contexts?

For studying SNRPC in disease contexts, researchers should consider multiple experimental models:

• Cellular models:

  • CRISPR/Cas9-engineered cell lines with SNRPC mutations or tagged endogenous SNRPC

  • Patient-derived induced pluripotent stem cells (iPSCs) differentiated into relevant cell types

  • Conditional knockout or knockdown systems to study acute versus chronic SNRPC loss

• Organismal models:

  • Drosophila models for high-throughput genetic interaction studies

  • Zebrafish for in vivo imaging of SNRPC dynamics and developmental splicing regulation

  • Mouse models with conditional tissue-specific SNRPC alterations

• In vitro disease models:

  • Reconstituted splicing systems with disease-associated SNRPC variants

  • Biomolecular condensate assays to evaluate phase separation properties under disease conditions

When selecting models, researchers should consider tissue-specific splicing requirements and potential compensatory mechanisms. For autoimmune conditions, humanized mouse models or patient-derived lymphocyte cultures can provide insights into how anti-SNRPC antibodies disrupt normal function. For neurodegenerative disease research, models should incorporate aging components and stress conditions that may precipitate condensate dysfunction .

How can high-throughput approaches be used to map the SNRPC interactome across different cellular conditions?

High-throughput approaches for mapping the dynamic SNRPC interactome should incorporate several complementary technologies:

• Proximity-based labeling methods:

  • BioID or TurboID fusions with SNRPC to identify proximal proteins under different conditions

  • APEX2-mediated labeling for capturing rapid, stimulus-dependent changes in the SNRPC interaction landscape

  • Split-proximity labeling for detecting specific SNRPC-partner interactions in distinct cellular compartments

• Mass spectrometry-based approaches:

  • Quantitative interaction proteomics using SILAC or TMT labeling to compare interactomes across conditions

  • Crosslinking mass spectrometry (XL-MS) to define interaction interfaces at amino acid resolution

  • Thermal proteome profiling to detect changes in SNRPC complex stability under different cellular states

• Genomics integration:

  • Integration of interactome data with transcriptomics to correlate SNRPC interactions with splicing outcomes

  • Combining ChIP-seq (for co-transcriptional splicing) with interactome analysis to map genome-wide SNRPC activity

  • Multi-omics data integration to construct predictive models of SNRPC function

These approaches should be applied across relevant cellular conditions including different cell cycle stages, stress responses, differentiation states, and disease models. The resulting datasets can be analyzed using machine learning approaches to identify condition-specific interaction modules and predict functional consequences of SNRPC complex remodeling .

What are the latest advances in understanding SNRPC's role in Tudor domain-mediated biomolecular condensation?

Recent advances have revealed that Tudor domains, including those in proteins that interact with SNRPC, represent a novel mechanism for biomolecular condensation through recognition of post-translational modifications . The SMN Tudor domain, in particular, has been shown to form condensates when bound to dimethylated arginine residues on protein ligands, establishing a new paradigm for how folded domains can drive phase separation . This mechanism contrasts with the traditional view that primarily intrinsically disordered regions mediate condensate formation.

SNRPC appears to participate in this process through its interaction with Tudor domain-containing proteins and potentially through its own post-translational modifications. The discovery that this property is shared among other Tudor domains suggests a general mechanism that may extend to various nuclear bodies and RNP granules . Current research is focusing on:

• Mapping the specific methylation sites on SNRPC that mediate Tudor domain interactions

• Characterizing how RNA binding influences the phase separation properties of these complexes

• Investigating how disease-associated mutations affect condensate formation and dynamics

• Developing small molecule modulators of Tudor domain interactions as potential research tools

These advances provide new perspectives on nuclear body assembly and function, with potential implications for understanding both normal cellular processes and pathological aggregation in diseases .

How might novel structural biology techniques advance our understanding of SNRPC dynamics and function?

Emerging structural biology techniques offer unprecedented opportunities to understand SNRPC dynamics and function:

• Cryo-electron tomography (cryo-ET) with focused ion beam (FIB) milling:

  • Enables visualization of SNRPC within native cellular environments

  • Provides structural insights into SNRPC organization within nuclear bodies

  • Allows correlation between SNRPC localization and functional nuclear architecture

• Integrative structural biology approaches:

  • Combining multiple data sources (X-ray crystallography, NMR, cryo-EM, crosslinking MS) to generate comprehensive structural models

  • Time-resolved structural studies to capture SNRPC conformational changes during splicing

  • Computational approaches like AlphaFold2 to predict structures of SNRPC complexes

• Single-molecule techniques:

  • smFRET (single-molecule Förster resonance energy transfer) to monitor SNRPC conformational dynamics

  • Optical tweezers combined with fluorescence to study force-dependent SNRPC-RNA interactions

  • Direct visualization of individual splicing events using zero-mode waveguides or nanopore approaches

These techniques can help resolve long-standing questions about SNRPC function, including:

• How SNRPC structural dynamics contribute to splice site recognition

• The structural basis for SNRPC participation in biomolecular condensates

• Conformational changes in SNRPC during spliceosome assembly and catalysis

• Structural effects of disease-associated modifications or mutations

By combining these approaches with functional assays, researchers can develop a mechanistic understanding of SNRPC's role in splicing regulation and nuclear body organization .