Recombinant Danio rerio Ubiquitin-like protein 5 (ubl5)

What is UBL5 and how does it differ from other ubiquitin-like proteins?

UBL5 is a highly conserved ubiquitin-like protein that possesses several unique structural features distinguishing it from other members of the UBL family. Unlike typical ubiquitin proteins, UBL5 lacks the C-terminal di-Gly motif necessary for forming covalent conjugates with other proteins. Instead, UBL5 contains a C-terminal dityrosine motif of unknown function . This structural difference suggests that UBL5 functions as a reversible regulator of protein functions rather than a protein degrader . The zebrafish UBL5 shares these distinctive features, making it an interesting model for studying non-canonical ubiquitin-like protein functions.

What are the known cellular functions of UBL5 in vertebrate systems?

UBL5 plays multiple critical roles in cellular function across vertebrate systems, which likely extend to Danio rerio:

• Pre-mRNA splicing regulation: UBL5 associates primarily with spliceosomal proteins and is essential for efficient pre-mRNA splicing . Depletion of UBL5 leads to decreased splicing efficiency and enhanced intron retention throughout the transcriptome .

• Sister chromatid cohesion maintenance: UBL5 is required for proper chromosome alignment and segregation during cell division . Loss of UBL5 function causes premature separation of sister chromatids, indicating its essential role in the fidelity of chromosome segregation .

• Endoplasmic reticulum stress response: UBL5 functions as an ER stress-responsive protein that undergoes rapid depletion in response to ER stressors . This depletion occurs through proteasome-dependent yet ubiquitin-independent proteolysis .

• Cell survival regulation: UBL5 serves as a physiologically relevant survival regulator. Knockdown of UBL5 activates multiple death pathways and induces severe apoptosis, while overexpression of UBL5 specifically protects against ER stress-induced apoptosis .

How evolutionarily conserved is UBL5 across species?

UBL5 demonstrates remarkable evolutionary conservation across species. The fundamental functions of UBL5 have been preserved from yeast to humans, particularly its role in pre-mRNA splicing. The yeast ortholog Hub1 promotes spliceosome functions similar to human UBL5, indicating an ancient and essential role in eukaryotic cellular processes . This high degree of conservation suggests that findings from various model organisms, including zebrafish, likely have broad biological relevance. The conservation pattern makes zebrafish UBL5 an excellent model for studying fundamental aspects of UBL5 biology that apply across vertebrate species.

What are the recommended methods for expressing and purifying recombinant Danio rerio UBL5?

Based on successful approaches with mammalian UBL5, the following methodology is recommended for zebrafish UBL5:

Expression System Selection:

• E. coli: BL21(DE3) strain with pET vector systems are suitable for high-yield expression

• Consider using a His-tag system for efficient purification as demonstrated in studies of human UBL5

Optimal Expression Conditions:

• Induction with 0.5 mM IPTG

• Expression at lower temperatures (16-18°C) overnight to enhance soluble protein yield

• Supplementation with 0.1 mM ZnCl₂ may improve proper folding

Purification Protocol:

• Harvest cells and lyse in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Purify using nickel affinity chromatography

• Apply size exclusion chromatography (Superdex 75) for higher purity

• For functional studies, consider tag removal using TEV protease

Quality Control Measures:

• SDS-PAGE to confirm purity (expected molecular weight ~8.5 kDa)

• Mass spectrometry to verify intact protein sequence

• Circular dichroism to assess proper folding

How should experiments be designed to study UBL5's role in pre-mRNA splicing in zebrafish?

When designing experiments to investigate zebrafish UBL5's role in pre-mRNA splicing, researchers should consider:

Knockdown/Knockout Approaches:

• Morpholino antisense oligonucleotides for transient knockdown

• CRISPR-Cas9 for stable genetic modifications

• Conditional knockout systems to avoid early developmental lethality

Splicing Assessment Methods:

• RNA-Seq analysis to measure global intron retention events, as demonstrated in human cell studies

• RT-PCR validation of specific candidate transcripts showing altered splicing

• Minigene splicing assays to test specific intron processing efficiency

Interaction Studies:

• Co-immunoprecipitation followed by mass spectrometry to identify spliceosomal interacting partners in zebrafish, focusing on homologs of known human UBL5 interactors like SART1, PRPC8, and EFTUD2

• Proximity ligation assays to confirm interactions in situ

• Yeast two-hybrid screening to identify direct binding partners

Rescue Experiments:

• Complementation with wild-type versus mutant UBL5 to determine structure-function relationships

• Overexpression of downstream splicing factors to identify compensatory mechanisms

Controls:

• Include knockdown of known splicing factors for comparison

• Use both scrambled morpholinos and uninjected controls

• Implement rescue experiments with wild-type UBL5 to confirm specificity

What approaches are recommended for studying UBL5's role in ER stress response in zebrafish models?

To effectively investigate UBL5's involvement in ER stress response in zebrafish models:

ER Stress Induction Protocols:

• Treatment with tunicamycin (1-5 μg/ml) to inhibit N-linked glycosylation

• Thapsigargin (0.1-1 μM) to deplete ER calcium stores

• Exposure to DTT (1-2 mM) to disrupt disulfide bond formation

UBL5 Stability Assessment:

• Western blot analysis to monitor UBL5 protein levels following ER stress induction

• Cycloheximide chase assays to determine UBL5 protein half-life under normal versus ER stress conditions

• Proteasome inhibitors (MG132) to confirm proteasome-dependent degradation mechanisms

UPR Pathway Analysis:

• qRT-PCR for UPR markers (BiP, CHOP, XBP1 splicing)

• Phosphorylation status of PERK and eIF2α by western blot

• Luciferase reporter assays for ATF6 and XBP1 transcriptional activity

Experimental Design Considerations:

• Time-course experiments to capture early versus late UPR events

• Dose-response studies to establish appropriate stressor concentrations

• Zebrafish developmental stage selection (larvae versus adult tissues)

Transgenic Approaches:

• Generate UBL5-GFP fusion lines to monitor subcellular localization changes during ER stress

• Create inducible UBL5 overexpression lines to assess protective effects against ER stress-induced apoptosis

What techniques are most effective for identifying UBL5 interaction partners in zebrafish?

Based on successful approaches used with mammalian UBL5, the following comprehensive methodology is recommended:

Quantitative Proteomics Approaches:

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture) labeling combined with immunoprecipitation and mass spectrometry analysis, similar to the approach that successfully identified human UBL5 interactions with spliceosomal proteins

• BioID proximity labeling: Fusion of zebrafish UBL5 with BirA* biotin ligase to identify proximal proteins in living cells

• Crosslinking mass spectrometry (XL-MS) to capture transient interactions

Biochemical Validation Methods:

• Co-immunoprecipitation experiments with tagged recombinant UBL5

• GST pull-down assays with recombinant proteins to confirm direct interactions

• Size exclusion chromatography to identify stable complexes

In Vivo Interaction Detection:

• Bimolecular Fluorescence Complementation (BiFC) assays in zebrafish cells

• FRET-based approaches to measure protein-protein interactions in zebrafish embryos

• PLA (Proximity Ligation Assay) to detect interactions in fixed tissues

Data Analysis Considerations:

• Apply stringent statistical filtering (p-value <0.05 and fold change >2)

• Validate top candidates by reciprocal pulldowns

• Perform Gene Ontology analysis to identify enriched functional categories, similar to the approach that revealed spliceosome and ribonucleoprotein complex enrichment in human UBL5 interactome

How can researchers effectively analyze UBL5's impact on transcriptome-wide splicing in zebrafish?

For comprehensive analysis of UBL5's effect on splicing in zebrafish:

RNA-Seq Experimental Design:

• Use at least 3 biological replicates per condition

• Consider developmental time points relevant to the research question

• Include multiple UBL5 knockdown/knockout approaches to control for off-target effects

Bioinformatic Analysis Pipeline:

• Align RNA-Seq reads to zebrafish genome (GRCz11/danRer11)

• Quantify alternative splicing events: exon skipping, intron retention, alternative 5'/3' splice sites

• Calculate Percent Spliced In (PSI) values for all annotated exons

• Apply specialized splice junction analysis tools (rMATS, MAJIQ, or LeafCutter)

Validation Strategies:

• RT-PCR validation of top differentially spliced events

• Minigene constructs to test specific splice sites in controlled conditions

• Cross-species comparison with known UBL5-dependent splicing events

Functional Impact Assessment:

• Conduct GO enrichment analysis on genes with altered splicing

• Analyze protein domain impacts of altered splicing

• Examine potential introduction of premature termination codons and NMD targets

Data Presentation:

Note: This table presents hypothetical data based on patterns observed in human cells . Actual numbers would depend on experimental results.

What are the optimal approaches for studying UBL5's impact on cell survival and apoptosis in zebrafish?

To effectively investigate UBL5's role in cell survival and apoptosis in zebrafish models:

In Vivo Techniques:

• TUNEL assays in zebrafish embryos following UBL5 knockdown/knockout

• Acridine orange staining to detect apoptotic cells in live embryos

• Transgenic zebrafish lines expressing fluorescent apoptosis reporters (secA5-YFP)

• Whole-mount immunostaining for cleaved caspase-3

Cellular Assays (using zebrafish cell lines):

• Flow cytometry with Annexin V/PI staining to quantify apoptotic populations

• Caspase activity assays (caspase-3/7, -8, -9) to delineate apoptotic pathways

• Mitochondrial membrane potential measurements (JC-1 or TMRE)

• Cell viability/proliferation assays (MTT, BrdU incorporation)

Stress Response Evaluation:

• Challenge with ER stressors (tunicamycin, thapsigargin) and measure survival

• UBL5 overexpression rescue experiments under stress conditions

• Time-course analysis of UBL5 protein levels following stress induction

Mechanistic Investigations:

• RNA-Seq analysis to identify death pathways activated upon UBL5 depletion

• Western blot analysis for key apoptotic markers (Bcl-2 family, p53)

• Co-immunoprecipitation to identify anti-apoptotic binding partners

Quantitative Analysis Framework:

Note: This table presents hypothetical data based on patterns observed in mammalian cells . Actual numbers would depend on experimental results.

How should researchers interpret conflicting data between zebrafish UBL5 studies and findings from other model organisms?

When encountering discrepancies between zebrafish UBL5 data and results from other models:

Systematic Analysis Framework:

• Compare experimental conditions:

  • Developmental timing differences

  • Tissue-specific versus whole-organism effects

  • Acute knockdown versus genetic knockout consequences

  • Completeness of protein depletion

• Consider evolutionary adaptations:

  • Species-specific UBL5 binding partners

  • Compensatory mechanisms in different organisms

  • Paralog redundancy in teleost fish (which underwent genome duplication)

• Technical considerations:

  • Antibody specificity and validation methods

  • Knockdown/knockout efficiency and verification

  • Expression system differences in recombinant studies

• Reconciliation strategies:

  • Perform cross-species rescue experiments (e.g., human UBL5 in zebrafish models)

  • Use domain swapping to identify functional differences

  • Conduct comparative interactome analysis across species

• Biological significance assessment:

  • Determine if differences reflect fundamental biological divergence or experimental artifacts

  • Consider whether divergent functions represent species-specific adaptations or core conserved functions

What are common technical challenges in zebrafish UBL5 experiments and how can they be addressed?

Researchers working with zebrafish UBL5 may encounter several technical challenges:

Antibody Specificity Issues:

• Problem: Limited availability of zebrafish-specific UBL5 antibodies

• Solution: Generate custom antibodies against zebrafish UBL5; validate using knockout controls; use epitope tagging approaches (HA, FLAG) for recombinant protein detection

Developmental Lethality:

• Problem: Complete loss of UBL5 may cause early developmental arrest

• Solution: Use conditional knockout systems; temperature-sensitive mutants; tissue-specific CRISPR; dose-dependent morpholinos; study hypomorphic alleles

Protein Stability Challenges:

• Problem: UBL5 undergoes rapid degradation during ER stress

• Solution: Use proteasome inhibitors for stabilization; perform experiments under controlled stress conditions; optimize sample preparation and handling to minimize degradation

Off-Target Effects:

• Problem: Morpholino or CRISPR approaches may have unintended consequences

• Solution: Use multiple independent knockdown reagents; perform careful rescue experiments; include appropriate controls; validate with genetic mutants

Functional Redundancy:

• Problem: Other proteins may compensate for UBL5 loss

• Solution: Consider combinatorial knockdown approaches; examine acute versus chronic loss effects; analyze compensatory gene expression changes

RNA-Seq Analysis Challenges:

• Problem: Complex splicing patterns can be difficult to interpret

• Solution: Use specialized splicing analysis tools; increase sequencing depth; validate key events with RT-PCR; consider longer read sequencing technologies

What criteria should be used to evaluate whether a phenotype is specifically due to UBL5 dysfunction?

To establish phenotypic specificity to UBL5 dysfunction:

Essential Validation Criteria:

Evaluation Framework Table:

What are promising strategies for identifying the direct splicing targets of UBL5 in zebrafish?

To identify direct splicing targets of UBL5 in zebrafish:

Integrative Genomics Approaches:

• CLIP-seq (Cross-Linking Immunoprecipitation): Map direct RNA-protein interactions by UV-crosslinking UBL5 to its bound RNAs, followed by immunoprecipitation and sequencing

• RNA-seq with splice junction analysis: Compare specific splicing events in control versus UBL5-depleted conditions

• Targeted intron retention analysis: Focus on introns most affected by UBL5 depletion

Structural and Biochemical Approaches:

• In vitro splicing assays: Test candidate pre-mRNAs with purified spliceosomes containing or lacking UBL5

• RNA-protein binding assays: Assess direct binding of recombinant UBL5 to candidate RNA sequences

• Spliceosome complex purification: Isolate distinct spliceosomal complexes and determine UBL5 association patterns

Genetic Screening Approaches:

• Suppressor screens: Identify mutations that rescue UBL5 depletion phenotypes

• Synthetic interaction screens: Discover genes that show enhanced phenotypes when partially depleted alongside UBL5

• CRISPR-based screens: Target splicing regulators to identify those that phenocopy UBL5 loss

Candidate Gene Testing:
Based on human studies, the cohesion protection factor Sororin represents a high-priority candidate for detailed investigation, as its missplicing appears to be a critical consequence of UBL5 depletion

How can researchers investigate potential zebrafish-specific functions of UBL5?

To explore potential zebrafish-specific functions of UBL5:

Comparative Genomics Strategies:

• Sequence analysis: Identify zebrafish-specific motifs or domains within UBL5

• Interactome comparison: Perform comparative proteomics across species to identify zebrafish-specific binding partners

• Expression pattern mapping: Characterize developmental and tissue-specific expression patterns unique to zebrafish

Specialized Zebrafish Approaches:

• Transparent embryo advantage: Utilize live imaging to track UBL5-GFP during development

• Tissue regeneration models: Investigate UBL5's role in zebrafish-specific regenerative processes (fin, heart, etc.)

• Temperature-sensitive experiments: Exploit poikilothermic nature of zebrafish to study temperature-dependent UBL5 functions

Unique Developmental Contexts:

• Maternal-to-zygotic transition: Examine UBL5's role during early developmental processes

• Metamorphosis: Investigate potential functions during larval-to-adult transition

• Circadian rhythm studies: Analyze UBL5's potential role in day/night-dependent processes

Zebrafish-Specific Applications:

• High-throughput screening: Develop zebrafish-based screens for compounds affecting UBL5 function

• Behavioral studies: Assess UBL5's impact on zebrafish-specific behaviors

• Environmental response: Examine UBL5's role in adaptation to environmental changes

What emerging technologies might advance zebrafish UBL5 research in the next five years?

Several cutting-edge technologies are poised to transform zebrafish UBL5 research:

Advanced Genome Editing Technologies:

• Prime editing: Enables precise genetic modifications without double-strand breaks

• Base editing: Allows specific nucleotide substitutions for subtle UBL5 modifications

• Inducible CRISPR systems: Permits temporal and spatial control of UBL5 editing

Single-Cell Technologies:

• Single-cell RNA-seq: Reveals cell type-specific UBL5 functions and splicing patterns

• Single-cell proteomics: Maps UBL5 protein interactions at cellular resolution

• Spatial transcriptomics: Preserves tissue context while analyzing UBL5-dependent gene expression

Advanced Imaging Methodologies:

• Super-resolution microscopy: Visualizes UBL5 subcellular localization at nanometer scale

• Light-sheet microscopy: Enables whole-organism imaging of UBL5 dynamics

• Optogenetic tools: Allows precise spatiotemporal control of UBL5 function

Integrative Multi-Omics Approaches:

• Integrated proteogenomics: Correlates UBL5-dependent transcriptome and proteome changes

• Metabolomics integration: Links UBL5 function to metabolic pathways

• Systems biology modeling: Predicts UBL5's role in complex cellular networks

Novel Protein Interaction Technologies:

• Proximity-dependent biotin identification (BioID): Maps UBL5's protein neighborhood in living cells

• APEX2 proximity labeling: Provides temporal resolution of UBL5 interactions

• MS-CETSA (Cellular Thermal Shift Assay): Assesses UBL5 protein interactions in living cells

The integration of these emerging technologies will likely provide unprecedented insights into the molecular functions and biological significance of UBL5 in zebrafish development, physiology, and disease models.