Recombinant U1 small nuclear ribonucleoprotein C (PY01791)

What is the structural composition of U1-snRNP and where does U1-C fit within this complex?

U1-snRNP is a complex ribonucleoprotein particle consisting of U1 snRNA and multiple protein components. The major protein components include U1-70K, U1-A, and U1-C, along with the Sm proteins that form a ring-like structure around the Sm site of the U1 snRNA. Unlike U1-70K and U1-A, which directly bind to stem-loops I and II of U1 snRNA respectively, U1-C does not interact directly with naked U1 snRNA but depends on other U1 protein components for association with the snRNP .

The U1-C protein contains a zinc finger-like motif in its N-terminal region (approximately within amino acids 7-47), which is important for its function. This region of U1-C has been shown to stimulate formation or stabilization of spliceosomal complex E, demonstrating its critical role in spliceosome assembly . Functionally, U1-C plays a vital role in recognizing the 5' splice site (5' ss) sequence, an activity that likely precedes and facilitates base pairing of U1 snRNA with the 5' splice site .

What specific domains and motifs are important for U1-C protein function?

The U1-C protein contains several functional domains that are crucial for its activity within the U1-snRNP complex:

• N-terminal region (amino acids 7-47): This region includes the zinc finger motif and homodimerization domain, which are essential for U1-C's function in spliceosome assembly .

• Zinc finger-like motif: Located within the N-terminal region, this motif contributes to U1-C's ability to recognize the 5' splice site sequence .

• Protein interaction domains: The N-terminal region mediates interactions with other proteins, including TIA-1 (T-cell intracellular antigen-1), which binds to U1-C to promote recruitment of U1-snRNP to specific 5' splice sites .

These structural elements are important considerations when working with recombinant U1-C protein, as mutations or truncations affecting these domains may significantly alter the protein's functionality in experimental settings.

How does recombinant U1-C protein interact with other components of the splicing machinery?

Recombinant U1-C protein participates in multiple protein-protein interactions that are crucial for splicing regulation:

• Interaction with U1-70K and Sm proteins: The N-terminal 45 amino acids of U1-C, which include the zinc finger-like motif, mediate interactions with U1-70K and the Sm proteins B'/B . These interactions are essential for U1-C's association with the U1-snRNP complex.

• Interaction with TIA-1: U1-C directly interacts with the splicing regulator TIA-1, specifically through its N-terminal region (amino acids 7-47) . This interaction is important for facilitating recruitment of U1-snRNP to certain 5' splice sites, particularly those with weak consensus sequences followed by uridine-rich stretches .

• Recognition of 5' splice site: U1-C can be cross-linked to the 5' splice site region and is responsible for the initial recognition of the 5' splice site sequence. This recognition event likely precedes and/or facilitates base pairing of U1 snRNA with the 5' splice site .

Understanding these interactions is crucial when designing experiments involving recombinant U1-C, as they may influence experimental outcomes in splicing assays or protein-protein interaction studies.

What experimental approaches are most effective for studying U1-C protein interactions in vitro?

Several experimental approaches have proven effective for studying U1-C protein interactions:

• Pull-down assays: GST-fusion proteins of interaction partners (e.g., TIA-1) can be used to pull down recombinant U1-C or its mutant derivatives . This approach has successfully demonstrated the specific interaction between U1-C and the glutamine-rich (Q) domain of TIA-1.

• Co-precipitation experiments: These have been valuable for defining the domains involved in protein-protein interactions. For example, studies have shown that the Q-rich domain of TIA-1 is necessary and sufficient for binding to U1-C, with this interaction enhanced by the RRM1 domain of TIA-1 .

• Domain mapping with deletion mutants: By testing various deletion mutants of U1-C (e.g., ΔN30, ΔN47), researchers have identified that amino acids between positions 7 and 47 are important for interaction with TIA-1 .

• Point mutation analysis: Introduction of point mutations in functional motifs (e.g., zinc finger motif) can help determine whether specific structural features are essential for protein-protein interactions .

• Cross-linking studies: These have been used to demonstrate that U1-C can be cross-linked to the 5' splice site region, supporting its role in 5' splice site recognition .

When designing such experiments, it's important to consider that some protein-protein interactions may be influenced by RNA binding or may require specific structural contexts.

How is U1-C involved in autoimmune diseases, and what experimental models best capture this pathophysiology?

U1-snRNP, including the U1-C component, is a target of autoreactive B cells and T cells in several rheumatic diseases, particularly systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) . The autoimmune response against U1-snRNP may be initiated and perpetuated through several mechanisms:

• T-cell epitope recognition: Specific epitopes within the U1-snRNP complex, including those in U1-70K and U1-A, have been identified as targets of autoreactive T cells . These epitopes can stimulate T-cell proliferation in vitro and induce epitope spreading in vivo.

• Epitope spreading mechanisms: Initially focused autoimmunity against one component can spread to other components of the U1-snRNP complex through several mechanisms:

  • Sequence homology between components containing RNA binding motifs (RNP1)

  • T-cell help provided to B cells recognizing different components of the same complex

  • B-cell internalization and presentation of new epitopes to T cells

Several experimental models have been used to study U1-snRNP autoimmunity:

• MRL/lpr mice: These mice develop autoimmunity against U1-snRNP components and exhibit T-cell reactivity to specific peptides from U1-70K and U1-A .

• NZB×NZW F1 mice: Administration of peptides from U1-70K to these mice, which do not typically produce autoantibodies against U1-snRNP, can induce T-cell reactivity and autoantibody production .

• T-cell clones from patients: T-cell clones generated from CTD patients have been useful for defining T-cell epitopes, studying cytokine production, and determining the frequency of autoreactive T cells .

What specific techniques can be used to assess the functional impact of U1-C mutations on splicing efficiency?

To assess how mutations in U1-C affect splicing efficiency, researchers can employ several techniques:

• In vitro splicing assays: Using cell-free extracts supplemented with recombinant wild-type or mutant U1-C protein to measure splicing of pre-mRNA substrates.

• Reconstitution experiments: Depleting endogenous U1-snRNP from extracts and reconstituting with purified components including wild-type or mutant U1-C protein .

• Spliceosome assembly assays: Monitoring the formation of splicing complexes (particularly complex E) in the presence of wild-type or mutant U1-C protein .

• 5' splice site recognition assays: Assessing the ability of U1-C variants to recognize and bind to 5' splice site sequences, which precedes base pairing with U1 snRNA .

• Minigene splicing assays: Transfecting cells with minigene constructs along with expression vectors for wild-type or mutant U1-C to assess splicing patterns in vivo.

When analyzing results from these assays, it's important to consider that:

• Different mutations may affect distinct aspects of U1-C function

• Some mutations might disrupt protein-protein interactions without affecting RNA recognition, or vice versa

• Certain splice sites may be more sensitive to U1-C mutations than others, particularly those with weak consensus sequences that rely heavily on auxiliary factors like TIA-1

What are the potential therapeutic implications of targeting U1-C in autoimmune disorders?

Understanding the interactions between U1-snRNP (including U1-C) and the immune system provides insights into potential therapeutic strategies for autoimmune disorders like SLE and MCTD :

• Targeted immunomodulation: By identifying specific T-cell epitopes within U1-C and other U1-snRNP components, it may be possible to develop targeted approaches to modulate autoreactive T-cell responses without broadly suppressing immune function.

• Disruption of epitope spreading: Interventions that interrupt the epitope spreading process could prevent progression of autoimmunity. This might involve targeting "driver T-cell clones" that express high-affinity TCRs and produce pro-inflammatory cytokines .

• Interference with TLR activation: U1-snRNA can activate Toll-like receptors (TLRs), contributing to the immunogenicity of the U1-snRNP complex . Strategies to block this activation pathway could reduce inflammation and autoantibody production.

• Inhibition of specific protein-protein interactions: As U1-C interacts with multiple partners, including TIA-1 , designing small molecules that specifically disrupt pathological interactions while preserving normal splicing function could represent a novel therapeutic approach.

• Modulation of apoptosis-induced modifications: Modifications to U1-snRNP components during apoptosis may enhance their immunogenicity . Interventions that prevent these modifications or their recognition by the immune system could reduce autoantibody production.

When developing such therapeutic strategies, it's essential to consider that U1-C plays a fundamental role in normal RNA splicing, so interventions must be carefully designed to target pathological processes while minimizing disruption of normal cellular functions.

How can recombinant U1-C protein be effectively expressed and purified for experimental use?

The successful expression and purification of recombinant U1-C protein involves several methodological considerations:

• Expression systems:

  • E. coli: Commonly used but may require optimization due to the presence of zinc finger motifs in U1-C.

  • Insect cells: May provide better folding and post-translational modifications for functional studies.

  • Mammalian cells: Most suitable for studies requiring native-like protein conformation and modifications.

• Expression constructs:

  • Fusion tags: GST, His, or MBP tags can facilitate purification and potentially enhance solubility.

  • Domain considerations: Full-length U1-C versus specific domains (e.g., N-terminal region containing the zinc finger motif).

  • Codon optimization: May be necessary for efficient expression in the chosen host system.

• Purification strategies:

  • Affinity chromatography: Using the fusion tag for initial capture.

  • Ion exchange chromatography: For further purification based on U1-C's charge properties.

  • Size exclusion chromatography: As a final polishing step to ensure homogeneity.

• Quality control:

  • SDS-PAGE and Western blotting: To confirm protein identity and purity.

  • Mass spectrometry: For precise molecular weight determination and identification of any modifications.

  • Functional assays: To verify that the purified protein retains its ability to interact with known partners or incorporate into U1-snRNP complexes.

• Storage considerations:

  • Buffer composition: Typically requires reducing agents and may benefit from zinc supplementation for stability of the zinc finger motif.

  • Storage temperature: Usually -80°C for long-term storage, with minimization of freeze-thaw cycles.

How can researchers effectively analyze the interaction between TIA-1 and U1-C in splicing regulation?

The analysis of TIA-1 and U1-C interactions in splicing regulation requires a multifaceted approach:

• Biochemical interaction studies:

  • Pull-down assays: Using GST-fusion proteins of TIA-1 domains to pull down U1-C .

  • Co-immunoprecipitation: From cell extracts expressing tagged versions of both proteins.

  • Domain mapping: Testing various deletion mutants and point mutations to identify critical interaction regions .

• Functional splicing assays:

  • In vitro splicing: Using pre-mRNAs with weak 5' splice sites followed by uridine-rich sequences (TIA-1 binding sites).

  • Minigene assays: To assess splicing patterns in cells with manipulated levels of TIA-1 or U1-C.

  • RNA-protein crosslinking: To detect direct interactions with pre-mRNA in cellular contexts.

• Structural studies:

  • NMR or X-ray crystallography: To characterize the physical interaction between the Q-rich domain of TIA-1 and the N-terminal region of U1-C .

  • Cryo-electron microscopy: To visualize the arrangement of proteins within the context of the U1-snRNP complex .

Table 1. Key domains involved in the TIA-1 and U1-C interaction

• Cell-based approaches:

  • RNA interference: To knock down TIA-1 or U1-C and assess effects on splicing patterns.

  • Overexpression studies: With wild-type or mutant proteins to identify dominant-negative effects.

  • CLIP (Cross-linking and immunoprecipitation): To identify the RNA targets of these proteins in vivo.

• Data analysis considerations:

  • Alternative splicing events should be quantified using methods like RT-PCR or RNA-seq.

  • Controls should include mutations in the TIA-1 binding site and in the 5' splice site.

  • The effect of TIA-1/U1-C interaction on splicing efficiency versus splice site selection should be distinguished.

What are the best methods for investigating the role of U1-C in T-cell epitope recognition and autoimmunity?

Investigating U1-C's role in T-cell epitope recognition and autoimmunity requires specialized immunological techniques:

• T-cell epitope identification:

  • Overlapping peptide screens: Synthesizing overlapping peptides spanning the U1-C sequence and testing their ability to stimulate T-cell proliferation .

  • MHC binding assays: To predict and confirm which peptides can bind to specific MHC molecules.

  • Elution of peptides from MHC molecules: Direct identification of naturally processed epitopes from antigen-presenting cells .

• T-cell response characterization:

  • T-cell proliferation assays: Measuring thymidine incorporation following in vitro stimulation with U1-C peptides .

  • Cytokine production analysis: Determining whether T cells produce Th1 (IFNγ, IL-2) or Th2 (IL-4, IL-10) cytokines in response to U1-C epitopes .

  • T-cell cloning: Generating and characterizing U1-C-specific T-cell clones from patients with autoimmune diseases .

• Animal models:

  • Immunization with U1-C peptides: To induce autoimmunity in non-autoimmune mice .

  • Transfer of U1-C-specific T cells: To determine their pathogenic potential.

  • Tracking epitope spreading: Monitoring the development of responses to additional epitopes following immunization with a single U1-C epitope .

• Human studies:

  • HLA association analysis: Determining whether certain HLA alleles are associated with responses to specific U1-C epitopes .

  • T-cell frequency determination: Estimating the frequency of U1-C-specific T cells in peripheral blood of patients versus healthy controls .

  • T-cell receptor (TCR) analysis: Characterizing the TCR repertoire of U1-C-specific T cells to identify potential "driver T-cell clones" .

• Therapeutic implications:

  • Tolerance induction: Testing whether administration of U1-C peptides under tolerogenic conditions can prevent or treat autoimmunity.

  • Blocking antibody approaches: Developing antibodies that mask key epitopes without affecting normal U1-C function.

  • Targeted immunomodulation: Designing strategies to specifically suppress pathogenic T-cell responses while preserving protective immunity.