Recombinant Chicken U1 small nuclear ribonucleoprotein C (SNRPC)

How should recombinant chicken SNRPC be expressed and purified for experimental use?

For expressing recombinant chicken SNRPC, an E. coli expression system similar to that used for human SNRPC would be appropriate. Based on protocols for human SNRPC, the protein can be expressed as a single, non-glycosylated polypeptide chain with a His-tag at the N-terminus to facilitate purification .

Expression Protocol:

• Clone the chicken SNRPC coding sequence into an appropriate expression vector with an N-terminal His-tag

• Transform into an E. coli expression strain (such as BL21(DE3))

• Induce protein expression with IPTG at optimal temperature and time conditions

• Harvest cells and lyse using appropriate buffer systems

Purification Methodology:

• Perform initial purification using Ni-NTA affinity chromatography

• Further purify using proprietary chromatographic techniques similar to those used for human SNRPC

• The purified protein should appear as a clear solution after sterile filtration

• Formulation can follow similar parameters to human SNRPC: protein solution containing buffer (such as Tris-HCl, pH 8.0), glycerol, NaCl, DTT, and EDTA

What are the optimal storage conditions for maintaining recombinant chicken SNRPC stability?

Based on stability guidelines for human SNRPC, the following storage recommendations would likely apply to chicken SNRPC:

• Short-term storage (2-4 weeks): Store at 4°C

• Long-term storage: Store frozen at -20°C

• To enhance stability during long-term storage, add a carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles as they could compromise protein integrity and activity

• Aliquot the protein solution before freezing to minimize freeze-thaw cycles

Proper storage is critical for maintaining the functional integrity of the protein, especially considering its role in complex molecular interactions within the spliceosome machinery.

How can chicken SNRPC be used to study evolutionary conservation of spliceosome function?

Chicken SNRPC provides an excellent model for comparative studies of spliceosome function across vertebrate species. Research approaches could include:

• Sequence alignment analysis: Compare amino acid sequences of chicken SNRPC with human and other vertebrate homologs to identify conserved functional domains and species-specific variations

• Functional complementation studies: Test whether chicken SNRPC can functionally replace human SNRPC in in vitro splicing assays to determine functional conservation

• Structural comparison: Analyze whether the three-dimensional structure of chicken SNRPC is similar to the human protein, particularly in regions known to interact with RNA and other spliceosomal proteins

The chicken U1 RNA gene enhancer has been shown to contain conserved DNA sequences spanning nucleotide positions -230 to -183 upstream of the transcriptional initiation site, which can be divided into distinct domains including an octamer sequence (ATGCAAAT) and an SPH domain . These regulatory elements may differ from human counterparts, suggesting species-specific regulation of U1 snRNP components.

What post-translational modifications occur in chicken SNRPC and how do they affect function?

While specific information on chicken SNRPC post-translational modifications (PTMs) is limited, insights can be drawn from human SNRPC studies:

• Expected PTMs: Based on human U1-C, the chicken homolog likely contains a C-terminal region rich in RG residues where arginines may undergo methylation

• Functional impact: These modifications likely affect protein-protein interactions within the spliceosome complex

• Research approach: Mass spectrometry analysis of native chicken SNRPC could identify specific PTMs and their positions

Research has shown that in human U1 snRNP, post-translationally modified C-terminal tails are responsible for the dynamics of U1-C and other components, and their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo . Similar regulatory mechanisms may exist in chicken SNRPC, making this an important area for comparative investigation.

How does chicken SNRPC interact with other components of the U1 snRNP complex?

Investigating the interactions of chicken SNRPC with other U1 snRNP components requires methodical approaches:

• Co-immunoprecipitation studies: Using antibodies against chicken SNRPC to pull down interacting partners

• Yeast two-hybrid screening: To identify direct protein-protein interactions

• In vitro reconstitution: Step-wise assembly of U1 snRNP components to determine order and requirements for SNRPC incorporation

Based on human U1-C studies, chicken SNRPC likely does not bind to free U1 snRNA but requires prior binding of Sm proteins and U1-70k . The zinc-finger region of SNRPC is probably critical for recognition of the 5' splice site by the U1 snRNP, similar to human U1-C. Differences in these interaction patterns between chicken and human could provide insights into species-specific aspects of spliceosome assembly and function.

What are the optimal in vitro assays for assessing chicken SNRPC function in splicing?

To investigate the functional activity of recombinant chicken SNRPC in splicing, researchers could employ the following methodologies:

• In vitro splicing assays:

  • Prepare chicken nuclear extracts depleted of endogenous SNRPC

  • Add recombinant chicken SNRPC at various concentrations

  • Assess splicing efficiency of reporter pre-mRNAs using RT-PCR or electrophoretic analysis

  • Compare activity with human SNRPC to identify species-specific functional differences

• 5' splice site recognition assays:

  • Use labeled RNA oligonucleotides containing consensus or variant 5' splice sites

  • Evaluate binding affinity and specificity of recombinant chicken SNRPC

  • Determine the contribution of SNRPC to splice site recognition through mutational analysis

• U1 snRNP assembly assays:

  • Reconstitute U1 snRNP particles using recombinant components

  • Analyze the requirements for incorporating chicken SNRPC into functional particles

  • Compare assembly pathways with human U1 snRNP assembly

How can structural studies of chicken SNRPC inform functional analysis?

Structural characterization of chicken SNRPC can provide valuable insights into its function:

• X-ray crystallography approaches:

  • Express and purify highly concentrated, homogeneous chicken SNRPC

  • Attempt crystallization alone or in complex with interacting partners

  • Solve the structure and compare with human SNRPC crystal structures

• NMR spectroscopy:

  • Particularly useful for analyzing dynamic regions like the C-terminal tail

  • Can provide information about conformational changes upon binding to RNA or proteins

• Cryo-electron microscopy:

  • Suitable for visualizing chicken SNRPC within the entire U1 snRNP complex

  • Allows for comparison with human U1 snRNP structural models

The human U1 snRNP structure has been elucidated by X-ray crystallography, showing how U1-C interacts with other components . Similar studies with chicken SNRPC would reveal conservation and differences in structural arrangements that could explain functional variations.

What techniques are most effective for analyzing chicken SNRPC-RNA interactions?

To characterize the interactions between chicken SNRPC and RNA components:

• Electrophoretic mobility shift assays (EMSA):

  • Incubate labeled U1 snRNA or pre-mRNA substrates with recombinant chicken SNRPC

  • Analyze complex formation by native gel electrophoresis

  • Use competition assays to determine binding specificity

• UV crosslinking:

  • Identify direct contact points between SNRPC and RNA molecules

  • Map the RNA binding interface of chicken SNRPC

• CLIP-seq (Crosslinking Immunoprecipitation followed by sequencing):

  • For genome-wide identification of RNA targets in chicken cells

  • Provides information about the RNA sequence preferences of chicken SNRPC in vivo

• Surface plasmon resonance:

  • For quantitative measurement of binding affinities and kinetics

  • Compare chicken SNRPC binding parameters with those of human SNRPC

How can researchers address solubility issues with recombinant chicken SNRPC?

Recombinant expression of SNRPC may present solubility challenges that can be addressed with these strategies:

• Optimization of expression conditions:

  • Test different E. coli strains (BL21, Rosetta, Arctic Express)

  • Vary induction parameters (temperature, IPTG concentration, duration)

  • Use auto-induction media to achieve gradual protein expression

• Solubility enhancement approaches:

  • Express as fusion protein with solubility tags (MBP, SUMO, GST)

  • Add solubility enhancers to lysis buffer (non-ionic detergents, increased salt concentration)

  • Consider co-expression with molecular chaperones

• Refolding strategies if expressed in inclusion bodies:

  • Solubilize inclusion bodies using appropriate denaturants

  • Perform step-wise dialysis to remove denaturants and allow proper refolding

  • Add stabilizing agents during refolding (L-arginine, glycerol)

Human SNRPC recombinant protein is typically formulated in buffer containing glycerol, NaCl, DTT, and EDTA , which suggests similar formulation may enhance chicken SNRPC solubility.

What controls should be included in functional studies of recombinant chicken SNRPC?

Robust controls are essential for validating functional studies:

• Positive controls:

  • Purified human SNRPC as a functional reference

  • Native chicken U1 snRNP complex isolated from chicken cells

• Negative controls:

  • Heat-denatured chicken SNRPC to confirm activity loss

  • Mutated chicken SNRPC variants with alterations in critical functional domains

  • Mock purification samples from non-transformed E. coli

• Specificity controls:

  • Non-related RNA binding proteins to verify binding specificity

  • Competition assays with specific and non-specific RNA substrates

• Validation approaches:

  • Complementation experiments in SNRPC-depleted extracts

  • Antibody inhibition studies using anti-SNRPC antibodies

How can researchers distinguish between chicken SNRPC isoforms and their functional significance?

Studies of human U1 snRNP have revealed differential incorporation of protein isoforms and dynamic interactions of subunits . To investigate chicken SNRPC isoforms:

• Isoform identification:

  • Perform transcriptome analysis to identify alternative splice variants

  • Use mass spectrometry to detect protein isoforms from chicken cells

  • Clone and express different isoforms as recombinant proteins

• Functional characterization:

  • Compare biochemical properties of different isoforms

  • Assess the ability of each isoform to reconstitute splicing activity

  • Analyze tissue-specific expression patterns of different isoforms

• Post-translational modification analysis:

  • Identify specific PTMs associated with each isoform

  • Determine how PTMs affect function and interactions

  • Create phosphomimetic or methylation-deficient mutants to study PTM effects

Studies on human U1 snRNP show that interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo , suggesting similar regulatory mechanisms may exist for chicken SNRPC isoforms.

How can chicken SNRPC be utilized in comparative studies of spliceosome evolution?

Comparative studies using chicken SNRPC can provide valuable evolutionary insights:

• Phylogenetic analysis:

  • Compare SNRPC sequences across diverse vertebrate species

  • Identify highly conserved regions that may be critical for function

  • Map species-specific variations that might relate to differences in splicing regulation

• Functional conservation testing:

  • Cross-species complementation assays to test functional equivalence

  • Analysis of binding specificity to different RNA targets across species

  • Investigation of interchangeability of SNRPC within reconstituted spliceosomes

• Regulatory element comparison:

  • Analyze conservation of enhancer elements regulating SNRPC expression

  • Compare the octamer sequence and SPH domain found in chicken U1 RNA gene with those in other species

  • Investigate how differences in regulatory elements affect expression patterns

What novel insights can be gained from studying chicken SNRPC dynamics within the spliceosome?

Investigating the dynamics of chicken SNRPC can reveal important aspects of spliceosome function:

• Assembly and disassembly kinetics:

  • Real-time monitoring of chicken SNRPC incorporation into U1 snRNP

  • Analysis of factors affecting the stability of SNRPC association

  • Comparison with human SNRPC dynamics to identify conserved and divergent features

• Conformational changes during splicing:

  • FRET-based approaches to detect structural rearrangements

  • Single-molecule studies to observe individual steps in the splicing reaction

  • Investigation of how post-translational modifications affect conformational dynamics

• Interaction networks:

  • Comprehensive mapping of chicken SNRPC interactions throughout the splicing cycle

  • Identification of species-specific interaction partners

  • Analysis of how these interactions change during spliceosome assembly and catalysis

Human U1 snRNP studies have shown that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of U1-C and other components . Similar dynamic behaviors likely exist in chicken SNRPC and could contribute to understanding the broader principles of spliceosome function.

What are the implications of chicken SNRPC research for understanding splicing-related diseases?

While primarily a basic research tool, studies of chicken SNRPC may have broader implications:

• Mechanism insights relevant to disease:

  • Understanding fundamental splicing mechanisms conserved across species

  • Identification of critical residues that, when mutated in humans, could lead to disease

  • Comparative analysis to pinpoint regions most susceptible to pathogenic variation

• Therapeutic development approaches:

  • Using chicken SNRPC as an alternative model for testing splicing modulators

  • Development of in vitro systems incorporating chicken components for drug screening

  • Identification of conserved binding pockets that could be targeted therapeutically

• Agricultural applications:

  • Insights into species-specific splicing regulation in avian systems

  • Potential applications in poultry health and development research

  • Understanding of how splicing contributes to avian-specific traits and disease resistance