Recombinant Gecko japonicus Protein BUD31 homolog (BUD31)

What is BUD31 and what protein family does it belong to?

BUD31 is a member of the BUD31 (G10) protein family and functions as a nuclear protein. It contains a putative nuclear translocation sequence, an N-terminal acidic domain, and a cysteine-rich C-terminal domain with a putative zinc-finger structure. Based on its structural characteristics, BUD31 is believed to function as a nuclear regulator of transcription . BUD31 is known to be highly conserved across species, suggesting its fundamental importance in cellular processes. The protein plays a critical role in both precatalytic and catalytic spliceosomes, where it helps stabilize protein interactions with pre-mRNA molecules .

What are the known functions of BUD31 in cellular processes?

BUD31 primarily functions in the splicing of pre-mRNA and is crucial for both the first and second catalytic steps of this process. Research has demonstrated that BUD31 associates early with the precatalytic B complex and maintains this association through activated, catalytic, and recycling spliceosome stages . Additionally, BUD31 is required for efficient cross-linking of a ~25-kDa protein with exon sequences near the 5′ splice site of pre-mRNA substrates. In the absence of BUD31, there is a compromised progression toward first-step catalysis and an arrest of the second step, particularly at higher reaction temperatures . Furthermore, recent studies have revealed BUD31's involvement in alternative splicing regulation during cell differentiation and development, particularly in spermatogenesis .

What are the optimal storage and handling conditions for recombinant BUD31?

For optimal maintenance of recombinant BUD31 activity, researchers should store the protein 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 . To enhance stability during long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) to the solution. Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and function. The standard formulation for BUD31 protein solution (0.25mg/ml) contains 20mM Tris-HCl buffer (pH 8.0), 0.2M NaCl, 40% glycerol, and 2mM DTT . This formulation helps maintain the protein's native conformation and functional properties during storage.

How can I design immunoprecipitation experiments to study BUD31's interactions in spliceosomes?

Immunoprecipitation of BUD31 from in vitro splicing reactions or native splicing extracts can be performed using either anti-polyoma monoclonal antibodies (for BUD31 fused to a polyoma tag) immobilized on protein-G-Sepharose, or IgG-agarose for TAP epitope-tagged BUD31 protein . For investigating BUD31's association with snRNAs, researchers can use S100 extracts from TAP-tagged strains. Typically, 30 μl of S100 extract is added to 30 μl of IgG-agarose and used for bead binding in NET buffer (10 mm Tris-Cl, 0.5% Nonidet P-40, 1 mm EDTA) supplemented with varying concentrations of NaCl (50, 150, 250, or 500 mm) to determine optimal binding conditions. Immunoprecipitated snRNAs can then be analyzed by Northern blot using radiolabeled U1, U2, U4, U5, and U6 DNA fragments of equal specific activity as probes . This methodology allows for characterization of BUD31's dynamic interactions with different spliceosomal components.

What approaches can be used to investigate BUD31's role in alternative splicing?

To study BUD31's function in alternative splicing, researchers can employ both in vitro splicing assays and in vivo genetic approaches. For in vitro analysis, splicing reactions can be performed using extracts from wild-type and BUD31-depleted cells, followed by RT-PCR or RNA-seq to identify differential splicing patterns . For in vivo studies, conditional knockout models provide valuable insights, as demonstrated in mouse studies where tissue-specific deletion of BUD31 revealed its essential role in spermatogenesis . When designing such experiments, researchers should consider:

• Creating conditional knockout models using Cre-loxP systems (e.g., using tissue-specific Cre drivers like VASA-Cre or Stra8-GFPCre)

• Validating knockout efficiency through qPCR, immunoblotting, and immunofluorescence staining

• Analyzing phenotypic consequences through histological examination and functional assays

• Performing RNA-seq to identify genome-wide alternative splicing events affected by BUD31 deletion

This comprehensive approach allows for detailed characterization of BUD31's role in regulating alternative splicing in specific biological contexts.

How does BUD31 contribute to spliceosome assembly and function at the molecular level?

BUD31's contribution to spliceosome assembly and function is multifaceted. Research indicates that BUD31 initially associates with the precatalytic B complex and maintains its presence through activated, catalytic, and recycling spliceosomes . At the molecular level, BUD31 appears to stabilize protein interactions with pre-mRNA, particularly near the 5' splice site. Studies have shown that in the absence of BUD31, cross-linking of a ~25-kDa protein with exon sequences near the 5′ splice site is compromised, suggesting that BUD31 facilitates or stabilizes this interaction .

The temperature-sensitive nature of BUD31's function provides further insights into its molecular role. Splicing reactions conducted with extracts lacking BUD31 show compromised progression toward first-step catalysis and completely arrested second-step catalysis at higher temperatures . This indicates that BUD31 likely contributes to maintaining the structural integrity of the spliceosome under various cellular conditions, which is critical for catalytic efficiency. Further research using high-resolution structural studies would help elucidate the precise molecular mechanisms through which BUD31 modulates spliceosome assembly and catalysis.

What is the relationship between BUD31 dysfunction and developmental abnormalities?

Research on BUD31 knockout mice has revealed significant developmental consequences when this protein is depleted. Global deletion of BUD31 in mice results in viable animals but with notably decreased body size, suggesting a broad developmental impact . More specifically, BUD31-null mice exhibit significantly smaller testes with severe germ cell loss in the tubules, indicating a critical role in reproductive development .

Tissue-specific deletion studies have further illuminated BUD31's developmental functions. Germ cell-specific knockout of BUD31 (Bud31-vKO) results in complete absence of Gcna-positive cells in testes by postnatal day 10, and these mice are completely infertile . Similarly, spermatogonia-specific knockout (Bud31-sKO) leads to greatly reduced testis size and significant loss of meiotic cells . These findings demonstrate that BUD31 is essential for proper spermatogenesis, likely through its role in regulating alternative splicing during cell differentiation and development. The specific alternative splicing events mediated by BUD31 that are critical for germ cell development represent an important area for future research.

How can cross-species comparisons of BUD31 enhance our understanding of its conserved functions?

While the search results primarily focus on human and mouse BUD31 rather than Gecko japonicus BUD31 specifically, cross-species comparisons offer valuable insights into the protein's evolutionary conservation and functional significance. The BUD31 protein is highly conserved across species, suggesting fundamental importance in cellular processes.

Researchers investigating Gecko japonicus BUD31 should consider the following comparative approaches:

• Sequence alignment analysis between Gecko japonicus BUD31 and orthologs from other species to identify conserved domains and species-specific variations

• Functional complementation studies to determine if Gecko japonicus BUD31 can rescue phenotypes in BUD31-deficient cells from other species

• Comparative analysis of BUD31's role in species-specific developmental processes, particularly reproductive development

• Investigation of potential differences in alternative splicing regulation between species

Such comparative studies could reveal both the core conserved functions of BUD31 and any species-specific adaptations, enhancing our understanding of this protein's biological roles across evolutionary history.

What are the common challenges in producing and purifying recombinant BUD31, and how can they be addressed?

Producing and purifying recombinant BUD31 presents several technical challenges that researchers should anticipate. Based on the recombinant human BUD31 production methods, the protein is typically expressed in E. coli with an N-terminal His-tag to facilitate purification . Common challenges and their solutions include:

For optimal results, researchers should aim for >85% purity as determined by SDS-PAGE, similar to the standards for commercially available recombinant human BUD31 .

How can I validate the functionality of recombinant BUD31 in experimental systems?

Validating the functionality of recombinant BUD31 is crucial before using it in experimental systems. Several approaches can be employed:

• In vitro splicing assays: Compare splicing efficiency using extracts supplemented with recombinant BUD31 versus control extracts, particularly at higher temperatures where BUD31's function becomes more critical .

• Co-immunoprecipitation studies: Assess whether the recombinant BUD31 can successfully interact with known spliceosomal partners.

• Complementation assays: Test if the recombinant protein can rescue splicing defects in BUD31-depleted cell extracts.

• Cross-linking experiments: Verify if recombinant BUD31 facilitates the cross-linking of the ~25-kDa protein to exon sequences near the 5′ splice site, which is a known function of endogenous BUD31 .

• Alternative splicing analysis: Evaluate if recombinant BUD31 can restore normal alternative splicing patterns in BUD31-deficient systems.

These validation approaches ensure that the recombinant BUD31 retains the functional properties of the native protein and is suitable for the intended research applications.

What controls should be included when analyzing BUD31's impact on alternative splicing?

When investigating BUD31's effect on alternative splicing, several controls are essential to ensure reliable and interpretable results:

• Positive controls: Include genes/transcripts known to be affected by BUD31 depletion, such as those identified in previous studies of BUD31 knockout mice .

• Negative controls: Analyze genes with constitutive splicing patterns that are unlikely to be affected by BUD31.

• Loading controls: When performing Western blots to confirm BUD31 knockdown/knockout, use ubiquitously expressed proteins like beta-actin as loading controls .

• Rescue experiments: Complement BUD31-depleted systems with wild-type BUD31 to demonstrate that observed splicing defects are directly due to BUD31 absence rather than off-target effects.

• Temperature controls: Since BUD31's function is temperature-sensitive, perform splicing reactions at multiple temperatures to fully characterize its role .

• Tissue-specific controls: When analyzing tissue-specific effects (e.g., in spermatogenesis), include appropriate tissue-specific markers (e.g., DAZL, Gcna for germ cells; Sox9 for Sertoli cells) to accurately interpret cell type-specific effects .

Incorporating these controls ensures robust experimental design and facilitates accurate interpretation of BUD31's role in alternative splicing regulation.

How might BUD31's role in alternative splicing connect to its potential functions in disease processes?

While the search results don't explicitly discuss BUD31's role in disease, its function in alternative splicing regulation suggests potential implications for various pathological conditions. Alternative splicing dysregulation is associated with numerous diseases, including cancer, neurodegenerative disorders, and developmental abnormalities. Given BUD31's essential role in spermatogenesis and developmental processes , dysfunction of this protein could potentially contribute to reproductive disorders and developmental pathologies.

Future research directions should explore:

• BUD31 expression patterns and potential mutations in patient samples from diseases associated with splicing dysregulation

• The specific alternative splicing events regulated by BUD31 that might be relevant to disease progression

• Potential therapeutic approaches targeting BUD31 or its downstream pathways in relevant disease contexts

Understanding these connections could provide new insights into disease mechanisms and identify novel therapeutic targets.

What technological advances could enhance our understanding of BUD31's function across different species?

Several emerging technologies could significantly advance our understanding of BUD31 function:

• CRISPR-Cas9 genome editing: Creating precise modifications in BUD31 across different species, including Gecko japonicus, would allow for detailed structure-function analyses and cross-species comparisons.

• Single-cell RNA sequencing: This technology could reveal cell type-specific alternative splicing events regulated by BUD31, particularly important in developmental contexts like spermatogenesis.

• Long-read sequencing technologies: These approaches provide a more comprehensive view of full-length transcripts, enabling better characterization of complex alternative splicing patterns influenced by BUD31.

• Cryo-electron microscopy: High-resolution structural studies of BUD31 within the spliceosome could elucidate its precise molecular interactions and mechanisms of action.

• Proteomics approaches: Advanced mass spectrometry techniques could identify the complete interactome of BUD31 across different cellular contexts and species.

These technological advances would provide unprecedented insights into BUD31's functions across evolutionary history and in various biological processes.

What are the most significant open questions regarding BUD31 function in cellular and developmental processes?

Despite significant advances in understanding BUD31, several critical questions remain unanswered:

• What are the specific alternative splicing events regulated by BUD31 during spermatogenesis and other developmental processes?

• How does BUD31 specifically stabilize protein-RNA interactions within the spliceosome?

• What are the tissue-specific functions of BUD31 beyond its role in spermatogenesis?

• How is BUD31 activity itself regulated in different cellular contexts?

• Are there species-specific differences in BUD31 function, particularly between mammals and reptiles like Gecko japonicus?

Addressing these questions will provide a more comprehensive understanding of BUD31's biological significance and potentially reveal new therapeutic targets for diseases associated with splicing dysregulation.

How can comparative studies between Gecko japonicus BUD31 and mammalian orthologs enhance evolutionary understanding?

Comparative studies between Gecko japonicus BUD31 and its mammalian orthologs represent a valuable approach for understanding the evolution of splicing regulation. By examining similarities and differences in protein sequence, expression patterns, and functional roles, researchers can identify:

• Core conserved domains that have maintained their function throughout evolutionary history

• Species-specific adaptations that may relate to unique aspects of reptile biology

• Evolutionary changes in BUD31's interaction network with other spliceosomal components

• Potential differences in alternative splicing regulation between reptiles and mammals