Recombinant Vitis vinifera U1 small nuclear ribonucleoprotein C (VIT_07s0104g01170)

What is the predicted functional role of U1 small nuclear ribonucleoprotein C in Vitis vinifera splicing?

Based on structural and functional studies of U1 snRNP in other organisms, the Vitis vinifera U1 small nuclear ribonucleoprotein C likely plays a crucial role in pre-mRNA splicing by helping stabilize the binding of U1 snRNP to the 5' splice site. The protein contains a zinc-finger domain that interacts with the duplex formed between pre-mRNA and the 5'-end of U1 snRNA . This interaction occurs through hydrogen bonds and electrostatic interactions with the RNA backbone around the splice junction rather than through base-specific contacts with pre-mRNA . The protein helps recruit U1 snRNP to weak 5' splice sites, participating in the early stages of spliceosome assembly . For functional characterization of Vitis vinifera U1-C, researchers should conduct binding assays with synthetic RNA duplexes mimicking grapevine 5' splice sites, followed by mutational analysis of key residues in the zinc-finger domain to confirm their role in RNA binding stabilization.

How can researchers determine the structure of Vitis vinifera U1-C and its interactions within the U1 snRNP complex?

To elucidate the structure of Vitis vinifera U1-C and its interactions, researchers should employ a multi-method approach. Begin with expressing and purifying the recombinant protein for crystallography trials, focusing on constructs containing the zinc-finger domain. For in-solution studies, isotopically label the protein (¹⁵N, ¹³C) for NMR spectroscopy to probe its dynamics and interactions with RNA duplexes . Reconstitution of the complete U1 snRNP particle from purified components would enable cryo-EM analysis to visualize the entire complex architecture . The specific methodology includes:

• Protein expression optimization:

  • Test multiple expression systems (bacterial, insect cell, plant-based)

  • Optimize solubility with fusion tags (MBP, SUMO) for the zinc-finger domain

  • Include zinc in expression media (10-50 μM ZnCl₂) to ensure proper folding

• Interaction mapping:

  • Co-precipitation experiments to identify direct protein-protein interactions

  • Focus on the interaction between U1-C and the N-terminal region of U1-70K, which is critical for stable association of U1-C with the complex

  • Use hydrogen-deuterium exchange mass spectrometry to map binding interfaces

What expression systems are optimal for producing functional recombinant Vitis vinifera U1-C protein?

For functional expression of Vitis vinifera U1-C, researchers should consider several expression systems, each with distinct advantages:

• Bacterial expression (E. coli):

  • Use BL21(DE3) or Rosetta strains to address potential codon bias

  • Express at low temperatures (16-18°C) to improve folding

  • Include zinc (10-50 μM ZnCl₂) in the growth medium

  • Optimal for isotopic labeling for NMR studies

  • Fusion tags (His₆-SUMO or MBP) can improve solubility

• Insect cell expression:

  • Baculovirus expression system provides eukaryotic post-translational modifications

  • Higher likelihood of proper folding for the zinc-finger domain

  • More suitable for complex formation with other U1 snRNP components

• Plant-based expression:

  • Transient expression in Nicotiana benthamiana

  • Native-like post-translational modifications

  • Appropriate for proteins that may require plant-specific chaperones

Purification strategy should include:

• Affinity chromatography based on fusion tag

• Heparin column for RNA-binding protein enrichment

• Ion exchange chromatography with high salt wash (500 mM NaCl) to remove nucleic acid contamination

• Size exclusion chromatography for final polishing

• Verification of zinc incorporation using atomic absorption spectroscopy

What are the key considerations for reconstituting functional Vitis vinifera U1 snRNP in vitro?

Reconstituting functional Vitis vinifera U1 snRNP requires careful attention to component preparation and assembly order. Based on established protocols for mammalian U1 snRNP , researchers should:

• Component preparation:

  • Express and purify all seven Sm proteins (SmB/B', SmD1, SmD2, SmD3, SmE, SmF, SmG)

  • Purify U1-specific proteins (U1-70K, U1-A, U1-C)

  • Synthesize U1 snRNA through in vitro transcription

• Assembly protocol:

  • First, form the Sm protein ring on the Sm site of U1 snRNA

  • Add U1-70K and U1-A proteins

  • Finally, incorporate U1-C, which requires U1-70K for stable association

• Optimal buffer conditions:

  • 20 mM HEPES-KOH pH 7.9

  • 100-150 mM KCl

  • 1.5-3 mM MgCl₂

  • 0.2 mM EDTA

  • 0.5 mM DTT

• Quality control:

  • Native gel electrophoresis to verify complex formation

  • Glycerol gradient centrifugation to separate fully assembled complexes

  • Functional testing through 5' splice site binding assays

  • Negative-stain electron microscopy to visualize particles

This in vitro reconstitution approach enables various structural and functional studies, including assessing the impact of mutations and examining interactions with splicing regulators .

How can researchers study the RNA binding properties of Vitis vinifera U1-C?

To characterize the RNA binding properties of Vitis vinifera U1-C, researchers should employ multiple complementary approaches:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Use fluorescently labeled RNA oligonucleotides containing 5' splice site sequences

  • Test binding of purified U1-C alone and within reconstituted U1 snRNP

  • Determine binding affinities for consensus and non-consensus splice sites

  • Compare binding to different RNA sequences to establish specificity profiles

• Filter binding assays:

  • Use 32P-labeled RNA oligonucleotides representing different 5' splice site sequences

  • Compare binding affinities with and without U1-C to determine its contribution

  • Analyze competition between wild-type and mutant splice sites

• Surface Plasmon Resonance (SPR):

  • Immobilize biotinylated RNA on streptavidin sensor chips

  • Measure binding kinetics (kon and koff) of U1-C and U1 snRNP

  • Determine the effect of mutations in the zinc-finger domain

• RNA binding specificity analysis:

  • Test whether U1-C stabilizes binding to non-consensus splice sites

  • Measure how U1-C affects the relative affinities of mismatched 5' splice sites

  • Determine if U1-C fine-tunes splicing specificity as observed in mammalian systems

Data analysis should include:

• Determination of dissociation constants (Kd)

• Hill coefficients to assess cooperativity

• Comparison between isolated U1-C and U1-C within the U1 snRNP complex

What approaches can be used to identify protein interaction partners of Vitis vinifera U1-C?

To comprehensively identify protein interaction partners of Vitis vinifera U1-C, researchers should implement multiple complementary techniques:

• Affinity purification coupled with mass spectrometry:

  • Express tagged U1-C (FLAG, HA, or TAP tag) in grapevine cell cultures

  • Perform pull-downs under different salt conditions to identify stable and transient interactions

  • Use cross-linking approaches to capture weak interactions

  • Analyze by LC-MS/MS with label-free quantification or SILAC

• Yeast two-hybrid screening:

  • Use U1-C as bait against a Vitis vinifera cDNA library

  • Screen for interactions with other splicing factors

  • Map interaction domains using truncated constructs

  • Validate interactions with co-immunoprecipitation

• Proximity-dependent biotin labeling (BioID or TurboID):

  • Fuse U1-C to a biotin ligase

  • Express in plant cells to label proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

  • Provides information about the native cellular environment

• Direct binding assays:

  • Focus on expected interactions with other U1 snRNP components

  • Test interactions with U1-70K, which is required for U1-C incorporation into U1 snRNP

  • Use purified recombinant proteins for in vitro binding studies

  • Quantify interactions using isothermal titration calorimetry or microscale thermophoresis

Data analysis should include:

• Filtering against appropriate negative controls

• Network analysis to identify interaction clusters

• Comparison with known interaction networks from model organisms

• Validation of key interactions through multiple methods

How can researchers assess the role of Vitis vinifera U1-C in alternative splicing regulation?

To investigate the role of Vitis vinifera U1-C in alternative splicing regulation, researchers should implement a comprehensive approach:

• Transcriptome-wide analysis:

  • Generate transgenic grapevine lines with modified U1-C expression (overexpression, knockdown, or knockout)

  • Perform RNA-seq with sufficient depth for alternative splicing detection

  • Analyze using specialized software (rMATS, SUPPA2, LeafCutter) to identify differential splicing events

  • Focus on changes in 5' splice site usage, particularly at non-consensus sites

• Minigene reporter assays:

  • Construct reporters containing alternatively spliced regions from grapevine genes

  • Include wild-type and mutated 5' splice sites

  • Express in grapevine protoplasts with normal or altered U1-C levels

  • Measure splicing outcomes using RT-PCR and quantitative analysis

• In vitro splicing assays:

  • Develop plant-specific in vitro splicing systems using grapevine nuclear extracts

  • Deplete endogenous U1-C and complement with recombinant protein

  • Test splicing of pre-mRNAs with consensus and non-consensus 5' splice sites

  • Compare splice site selection patterns with and without U1-C

• CLIP-seq (Cross-linking and Immunoprecipitation followed by sequencing):

  • Perform in vivo UV crosslinking of U1-C to bound RNAs

  • Immunoprecipitate U1-C-RNA complexes

  • Identify direct RNA binding sites genome-wide

  • Correlate binding patterns with alternative splicing outcomes

Data interpretation should focus on:

• Identifying splice sites particularly dependent on U1-C function

• Determining if U1-C preferentially affects weak 5' splice sites as in mammalian systems

• Classifying affected genes into functional categories to reveal biological impacts

What techniques can researchers use to study the functional interplay between U1-C and other splicing factors in Vitis vinifera?

To investigate functional interactions between U1-C and other splicing regulators in Vitis vinifera:

• Co-immunoprecipitation followed by functional assays:

  • Identify proteins that co-purify with U1-C from nuclear extracts

  • Focus on interactions with known splicing regulators (e.g., TIA-1, which interacts with U1-C in mammals )

  • Test whether co-expression of interacting factors alters splicing patterns

  • Perform domain mapping to identify critical interaction surfaces

• Genetic interaction studies:

  • Generate plants with altered expression of both U1-C and candidate interacting factors

  • Analyze splicing patterns to identify synergistic or antagonistic effects

  • Look for rescue of splicing defects by overexpression of interacting partners

  • Identify target genes co-regulated by multiple factors

• RNA-protein complexes characterization:

  • Use sequential immunoprecipitation to isolate complexes containing U1-C and other factors

  • Analyze RNA content by sequencing to identify co-regulated targets

  • Perform in vitro reconstitution with purified components

  • Test cooperative RNA binding using EMSA and filter binding assays

• Functional reconstitution experiments:

  • Deplete specific factors from nuclear extracts

  • Complement with recombinant proteins individually or in combination

  • Measure splicing efficiency and accuracy

  • Test assembly of splicing complexes by native gel electrophoresis

Data analysis should focus on:

• Identifying specific 5' splice sites co-regulated by multiple factors

• Determining whether interactions are direct or mediated by other components

• Mapping the sequential binding of factors during spliceosome assembly

• Developing comprehensive models of co-regulation networks

How do specific domains of Vitis vinifera U1-C contribute to its function in splicing?

To elucidate the domain-specific functions of Vitis vinifera U1-C, researchers should perform systematic structure-function analysis:

• Domain identification and characterization:

  • Identify the N-terminal zinc-finger domain, which is critical for RNA binding

  • Map potential protein interaction domains by sequence analysis

  • Predict post-translational modification sites

  • Generate structural models based on homology to characterized U1-C proteins

• Mutational analysis:

  • Generate domain deletion constructs (ΔZn-finger, ΔC-terminal)

  • Create point mutations in conserved residues of the zinc-finger domain

  • Express mutant proteins and test for:

    • RNA binding capacity

    • Protein-protein interactions

    • Incorporation into U1 snRNP

    • Splicing activity in complementation assays

• Functional complementation:

  • Generate U1-C-depleted splicing extracts

  • Add back wild-type or mutant U1-C proteins

  • Test splicing efficiency and accuracy

  • Analyze 5' splice site selection patterns

• Structural studies of domain-specific functions:

  • Determine structures of individual domains

  • Map RNA binding surfaces through NMR chemical shift perturbation

  • Identify protein interaction interfaces

  • Compare with structures from other organisms to identify conserved features

Expected results based on mammalian studies:

• The zinc-finger domain should be essential for RNA binding and splicing function

• U1-C likely requires interactions with U1-70K for incorporation into U1 snRNP

• The protein should stabilize the interaction between U1 snRNA and the 5' splice site through contacts with the RNA backbone

How do mutations in the zinc-finger domain affect the function of Vitis vinifera U1-C?

To characterize the effects of zinc-finger domain mutations on U1-C function:

• Targeted mutagenesis approach:

  • Identify conserved cysteine and histidine residues that coordinate zinc

  • Mutate these residues individually (C→S, H→A) or in combination

  • Target residues that interact with RNA based on mammalian U1-C structures

  • Create a panel of mutations with varying severity

• Functional analysis of mutants:

  • Test zinc binding capacity using spectroscopic methods

  • Assess protein folding and stability through circular dichroism

  • Measure RNA binding affinity using filter binding assays or SPR

  • Determine incorporation into U1 snRNP through co-immunoprecipitation

  • Evaluate splicing activity in complementation assays

• Analysis of splice site selection:

  • Test binding to consensus and non-consensus 5' splice sites

  • Compare effects on strong versus weak splice sites

  • Determine if mutations affect the ability of U1-C to fine-tune splice site selection

  • Use competition assays to measure relative affinities for different splice sites

• Structural characterization of mutants:

  • Obtain crystal structures or NMR data of mutant proteins

  • Compare structural changes with functional defects

  • Identify conformational changes that affect RNA binding or protein interactions

Expected outcomes based on published data:

• Mutations disrupting zinc coordination should abolish RNA binding and splicing function

• Some mutations may specifically affect U1-C's ability to stabilize binding to non-consensus splice sites

• Certain mutations might disrupt protein-protein interactions while preserving RNA binding

How does Vitis vinifera U1-C compare with its orthologs in other plant species?

To understand species-specific features of Vitis vinifera U1-C through comparative analysis:

• Comparative sequence analysis:

  • Align U1-C sequences from Vitis vinifera, Arabidopsis thaliana, Oryza sativa, and other plant species

  • Calculate sequence identity and similarity percentages for full-length proteins and functional domains

  • Identify grapevine-specific sequence features

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural conservation analysis:

  • Generate structural models of U1-C from different plant species

  • Compare the zinc-finger domains and RNA-binding surfaces

  • Identify conserved versus variable regions

  • Map conservation scores onto structural models

• Functional complementation experiments:

  • Express Vitis vinifera U1-C in Arabidopsis or rice U1-C mutants

  • Test ability to rescue splicing defects

  • Compare with complementation by native U1-C

  • Identify species-specific functions through domain swapping

• Splice site preference analysis:

  • Compare consensus 5' splice site sequences across plant species

  • Test whether Vitis vinifera U1-C has different sequence preferences than orthologs

  • Analyze how these differences correlate with genome-wide splice site distributions

Expected findings:

• The zinc-finger domain should show high conservation across plant species

• Species-specific variations might be found in protein interaction regions

• Functional complementation across species should be possible but may reveal subtle differences in splicing regulation

What insights can be gained by comparing plant and mammalian U1-C proteins?

Comparative analysis between plant and mammalian U1-C proteins can reveal both fundamental conservation and lineage-specific adaptations:

• Sequence and structural comparison:

  • Align Vitis vinifera U1-C with human and other mammalian orthologs

  • Compare domain organization and key functional residues

  • Identify plant-specific insertions or deletions

  • Generate structural models to visualize differences

• Cross-species functional studies:

  • Test whether plant U1-C can interact with mammalian U1 snRNP components

  • Examine if plant U1-C can recognize mammalian 5' splice sites

  • Determine if the fine-tuning role in splice site selection is conserved

  • Perform reciprocal complementation studies with mammalian U1-C in plant systems

• Analysis of binding mechanisms:

  • Compare RNA binding specificities between plant and mammalian proteins

  • Test whether plant U1-C makes similar contacts with the RNA backbone

  • Determine if plant U1-C also lacks base-specific contacts with pre-mRNA

  • Examine differences in interaction with non-consensus splice sites

• Hybrid complex formation:

  • Reconstitute U1 snRNP with mixed plant and mammalian components

  • Test functionality in splicing assays

  • Identify compatible and incompatible interactions

  • Determine minimal conserved features required for function

Expected outcomes:

• The zinc-finger domain should show functional conservation across kingdoms

• Differences may be observed in protein-protein interactions, particularly with U1-70K

• Basic mechanisms of splice site recognition should be conserved, but specificity may differ

• Plant-specific adaptations might relate to differences in splice site consensus sequences

How can structural knowledge of Vitis vinifera U1-C be applied to manipulate splicing patterns in grapevine?

Applying structural insights about U1-C to manipulate splicing in grapevine could open new avenues for crop improvement:

• Engineered U1-C variants for splicing modulation:

  • Design mutations that alter splice site preference

  • Create variants with enhanced or reduced activity

  • Target specific classes of splice sites

  • Express in transgenic plants to modify splicing patterns

• Splice-switching oligonucleotides (SSOs):

  • Design complementary to 5' splice sites

  • Develop SSOs that either block or enhance U1-C binding

  • Target specific genes for splicing modulation

  • Optimize chemistry for stability and delivery in plants

• Structure-based small molecule screening:

  • Identify binding pockets in the U1-C structure

  • Screen for compounds that modulate U1-C function

  • Develop splicing modulators for research tools

  • Target specific splicing events relevant to stress response or development

• CRISPR-based approaches:

  • Modify endogenous U1-C to alter function

  • Engineer 5' splice sites to modulate U1-C dependency

  • Create conditional U1-C variants for temporal control

  • Develop multiplexed approaches to target multiple splicing events

Methodology for validation:

• RNA-seq to monitor global splicing changes

• Targeted RT-PCR for specific splicing events

• Phenotypic analysis of modified plants

• Stress testing to evaluate impacts on plant resilience

What are the most promising directions for future research on Vitis vinifera U1-C?

Future research on Vitis vinifera U1-C should focus on several promising directions:

• Stress-responsive splicing regulation:

  • Investigate how U1-C function changes under drought, heat, or pathogen stress

  • Identify stress-specific protein modifications or interactions

  • Determine if U1-C is involved in stress-induced alternative splicing

  • Develop splicing-based markers for stress response

• Tissue-specific splicing regulation:

  • Compare U1-C expression and modification across tissues

  • Identify tissue-specific interacting partners

  • Map tissue-specific alternative splicing events dependent on U1-C

  • Correlate with developmental processes unique to grapevine

• Integration with other RNA processing mechanisms:

  • Study connections between U1-C and polyadenylation

  • Investigate roles in premature termination of transcription

  • Examine interactions with nonsense-mediated decay machinery

  • Map the complete RNA processing network involving U1-C

• Agricultural applications:

  • Identify splicing events affecting fruit quality

  • Study U1-C's role in disease resistance gene splicing

  • Develop U1-C variants optimized for different growing conditions

  • Create diagnostic tools based on splicing profiles

Experimental approach should include:

• Multi-omics integration (transcriptomics, proteomics, metabolomics)

• Development of grapevine-specific splicing assays

• Creation of tissue-specific and inducible U1-C variants

• Field testing of promising modifications

What are common issues in recombinant expression of U1-C and how can they be addressed?

Researchers frequently encounter challenges when expressing recombinant U1-C protein:

• Protein insolubility issues:

  • Problem: The zinc-finger domain often aggregates during expression

  • Solution: Express at lower temperatures (16-18°C), use solubility tags (SUMO, MBP), and include zinc (10-50 μM ZnCl₂) in the growth medium

  • Validation: Analyze soluble fraction by SDS-PAGE and Western blot

• Improper zinc incorporation:

  • Problem: Zinc-finger domain fails to fold properly without zinc

  • Solution: Add ZnCl₂ to expression media and purification buffers, maintain reducing environment (2-5 mM β-mercaptoethanol)

  • Validation: Measure zinc content by atomic absorption spectroscopy, analyze folding by circular dichroism

• RNA contamination:

  • Problem: Co-purification of bacterial RNA affects homogeneity

  • Solution: Include high salt washes (500 mM NaCl), treat with RNase A during purification, use heparin chromatography

  • Validation: Check A260/A280 ratio, analyze by native gel electrophoresis with and without RNase treatment

• Proteolytic degradation:

  • Problem: U1-C is sensitive to proteolysis during expression and purification

  • Solution: Use protease-deficient strains, include protease inhibitors throughout purification, minimize handling time

  • Validation: Analyze sample stability over time by SDS-PAGE, perform Western blot with antibodies against different epitopes

• Low expression yields:

  • Problem: Poor expression due to toxicity or codon bias

  • Solution: Use rare codon-optimized strains, optimize codon usage for expression host, use inducible promoters with tight regulation

  • Validation: Compare expression levels across various conditions and strains

Comprehensive optimization strategy:

• Test multiple construct designs with different boundaries and tags

• Screen multiple expression systems in parallel

• Develop robust purification protocols with quality control checkpoints

• Verify protein activity through functional assays

How can researchers troubleshoot functional assays for U1-C activity?

When functional assays for U1-C activity yield unexpected results, consider these troubleshooting approaches:

• RNA binding assays:

  • Problem: Weak or no binding detected in EMSA or filter binding assays

  • Troubleshooting: Verify protein integrity by mass spectrometry, test multiple buffer conditions, include positive controls, ensure RNA quality

  • Optimization: Try different RNA sequences, include competitor RNAs, optimize protein:RNA ratios

• In vitro splicing assays:

  • Problem: Low splicing efficiency or high background

  • Troubleshooting: Check extract quality, optimize salt and ATP concentrations, ensure even sample heating

  • Optimization: Use radiolabeled pre-mRNAs for higher sensitivity, include internal controls, extend reaction times

• Protein-protein interaction assays:

  • Problem: Failure to detect expected interactions

  • Troubleshooting: Verify protein expression by Western blot, test milder lysis conditions, include positive controls

  • Optimization: Try different tagging strategies, use crosslinking approaches, optimize buffer composition

• Reconstitution of U1 snRNP:

  • Problem: Incomplete assembly or inactive complexes

  • Troubleshooting: Analyze individual components for activity, optimize assembly order, verify RNA integrity

  • Optimization: Use stepwise assembly with validation at each step, purify fully assembled complexes by affinity chromatography

• In vivo splicing analysis:

  • Problem: No detectable splicing changes upon U1-C manipulation

  • Troubleshooting: Verify protein expression/knockdown efficiency, check for potential compensation mechanisms

  • Optimization: Use more sensitive detection methods, focus on specific target genes, analyze multiple splicing events

Validation approach:

• Include appropriate positive and negative controls

• Incorporate internal standards to normalize between experiments

• Test multiple assay formats to corroborate findings

• Verify key results through independent methodologies

What statistical approaches are appropriate for analyzing U1-C effects on alternative splicing?

Proper statistical analysis is crucial for interpreting U1-C's impact on alternative splicing:

• Differential splicing analysis:

  • Apply specialized tools like rMATS, SUPPA2, or LeafCutter

  • Calculate percent spliced in (PSI) values for alternative events

  • Use appropriate statistical tests (e.g., likelihood ratio test, Bayesian inference)

  • Implement false discovery rate (FDR) correction for multiple testing

• Quantification metrics:

  • For exon skipping: PSI (percent spliced in)

  • For intron retention: IR ratio (retained/spliced)

  • For alternative 5'/3' splice sites: relative usage of each site

  • For mutually exclusive exons: relative inclusion of each exon

• Experimental design considerations:

  • Include sufficient biological replicates (minimum 3, preferably more)

  • Control for batch effects using appropriate experimental design

  • Use paired analysis when comparing treatment and control from the same samples

  • Consider sequencing depth requirements (40-50M reads minimum for alternative splicing)

• Correlation analysis:

  • Correlate splicing changes with U1-C binding (from CLIP-seq)

  • Analyze relationship between splice site strength and U1-C dependency

  • Integrate with other datasets (expression, chromatin, other splicing factors)

  • Implement multivariate analysis to identify co-regulated events

Data visualization approaches:

• Sashimi plots for individual splicing events

• Volcano plots for global splicing changes

• Heatmaps for clustering related splicing events

• Sequence logos for motif analysis around affected splice sites

How should researchers interpret U1-C binding data in the context of splice site selection?

To properly interpret U1-C binding data and its relationship to splice site selection:

• Integration of binding and functional data:

  • Overlay U1-C binding sites (from CLIP-seq) with splicing changes (from RNA-seq)

  • Calculate enrichment of binding around regulated versus non-regulated splice sites

  • Determine positional preferences relative to 5' splice sites

  • Correlate binding strength with splicing efficiency

• Sequence context analysis:

  • Perform motif discovery around U1-C binding sites

  • Compare bound versus non-bound 5' splice sites

  • Analyze RNA secondary structure potential around binding sites

  • Identify features that distinguish U1-C-dependent splice sites

• Mechanistic interpretation:

  • Determine if U1-C primarily affects weak 5' splice sites as in mammals

  • Assess whether U1-C in grapevine, like in humans, lacks base-specific contacts

  • Evaluate if binding stabilizes U1 snRNP recruitment through interactions with the RNA backbone

  • Test if U1-C fine-tunes the relative affinities for various 5' splice sites

• Comparative analysis approach:

  • Compare U1-C binding patterns with those of U1 snRNA

  • Assess co-binding with other splicing factors

  • Analyze evolutionary conservation of binding sites

  • Compare binding profiles under different conditions (e.g., stress, development)

Visualization and interpretation tools:

• Genome browser tracks showing binding intensity and splicing changes

• Metagene plots centered on 5' splice sites

• RNA maps showing position-dependent effects

• Network analysis of co-regulated splicing events