BUD31 Human

What is BUD31 and what is its primary function in human cells?

BUD31 is a conserved spliceosomal component essential for spliceosome assembly and catalytic activity. Research demonstrates that BUD31 predominantly associates with U2 snRNPs and hnRNP proteins within the spliceosome complex . Functionally, BUD31 regulates alternative splicing (AS) by binding to intronic and exonic regions near splice sites, influencing exon inclusion/skipping and intron retention events .

Interaction studies using immunoprecipitation coupled with mass spectrometry reveal that BUD31 forms complexes with multiple spliceosome components, particularly those involved in early spliceosome assembly. In ovarian cancer cells, GO enrichment analysis confirmed significant enrichment for "mRNA splicing via the spliceosome" and "regulation of RNA splicing" pathways among BUD31-interacting proteins .

What experimental methods are most effective for studying BUD31's role in alternative splicing?

Multiple complementary methodologies are necessary for comprehensive analysis of BUD31's splicing functions:

• RNA-protein interaction mapping:

  • RNA immunoprecipitation sequencing (RIP-seq): Identifies genome-wide binding sites, revealing that BUD31 predominantly binds to intron regions (81.8%) of pre-mRNAs

  • SpyTag-based CLIP (SpyCLIP): Provides covalent link-based mapping of protein-RNA interactions with high efficiency and accuracy

• Alternative splicing detection:

  • SMART-seq: Reveals widespread exon-skipping and intron retention events upon BUD31 deletion

  • Fragment analysis and semi-quantitative RT-PCR: Validates specific splicing events using isoform-specific primers

• Functional validation:

  • Minigene splicing assays: Confirms direct regulation of target pre-mRNAs when combined with BUD31 siRNAs

  • Splicing reporter assays: Quantifies the effect of BUD31 on specific splicing events using luciferase reporters

• In vivo models:

  • Conditional knockout models (e.g., Bud31-sKO mice): Evaluate tissue-specific functions and physiological relevance

  • Xenograft models with inducible knockdown systems: Assess impact on tumor growth and metastasis

What are the RNA binding motifs recognized by BUD31?

BUD31 recognizes specific RNA sequence motifs that facilitate its splicing regulatory function. Research has identified distinct binding patterns:

• In ovarian cancer cells: HOMER algorithm analysis identified ACUUACCU as the most abundant 8-mer motif recognized by BUD31 . Interestingly, 2 of the 4 top-scoring motifs were located near the 5' splice site (5ss) intron-exon junction and were reverse complemented .

• In male germ cells: The most abundant elements were UUUUAAAA and GAGGCAGG motifs . CLIP-seq analysis demonstrated that BUD31 is highly enriched in exon-intron regions around splicing sites .

These binding preferences explain BUD31's ability to regulate specific subsets of alternative splicing events. Motif location analysis reveals that BUD31 binding sites are concentrated at exon-intron boundaries, consistent with its role in splice site selection during spliceosome assembly .

How does BUD31 regulate alternative splicing patterns genome-wide?

BUD31 has a profound impact on global splicing patterns, with distinct regulatory signatures:

• Types of splicing events regulated:

  • The predominant alternative splicing event upon BUD31 knockdown is exon skipping (68.0% of identified events)

  • Other events include retained introns, alternative 5' splice sites, alternative 3' splice sites, and mutually exclusive exons

• Effect on coding sequence length:

  • BUD31 knockdown significantly decreases the abundance of long coding sequence (CDS) isoforms (750-1750 bp)

  • Simultaneously increases the abundance of shorter CDS isoforms (100-650 bp)

  • The average CDS length of all isoforms decreases after BUD31 silencing

• Nonsense-mediated decay (NMD) consequences:

  • Analysis identified 6,325 NMD-sensitive splice isoforms affected by BUD31

  • Expression of genes acquiring increased NMD-sensitive isoform fractions decreases upon BUD31 knockdown

These findings indicate that BUD31 generally promotes exon inclusion and the production of longer functional protein isoforms across the transcriptome.

What are the tissue-specific functions of BUD31 in humans?

BUD31 exhibits distinct tissue-specific functions based on tissue context:

This tissue specificity likely derives from differential expression patterns, tissue-specific cofactors, and distinct substrate pre-mRNAs available in each cellular context.

What are the key splicing targets of BUD31 with functional significance?

Integrated analysis of BUD31-bound genes and alternatively spliced genes has identified several direct splicing targets with functional importance:

• CDK2 in male germ cells:

  • BUD31 deletion leads to retention of the first intron of CDK2 pre-mRNA

  • RIP-seq confirms direct binding of BUD31 to intron 1 of CDK2

  • BUD31 depletion increases intron-containing transcripts, reducing functional CDK2 protein

  • CDK2 is essential for male fertility, explaining one mechanism of the infertility phenotype

• BCL2L12 in ovarian cancer:

  • BUD31 promotes exon 3 inclusion to generate full-length BCL2L12 (BCL2L12-L), an anti-apoptotic protein

  • BUD31 knockdown promotes exon 3 skipping, generating a truncated isoform (BCL2L12-S)

  • The truncated isoform undergoes nonsense-mediated mRNA decay, reducing anti-apoptotic protection

• Additional validated targets:

  • PRDM8: BUD31 depletion leads to skipping of exon 5

  • CCNT1: BUD31 ablation results in inclusion of exon 7

  • GO analysis of BUD31 targets reveals enrichment in pathways related to apoptosis, cell cycle, splicing, and autophagy

Combined CLIP-seq and RNA-seq analysis revealed approximately 57% (1,408/2,465) of genes with alternative splicing events upon BUD31 knockdown were directly bound by BUD31 , indicating its broad but specific regulatory role.

How does BUD31 dysregulation contribute to cancer progression?

BUD31 functions as an oncogenic driver in ovarian cancer through several mechanisms:

These findings collectively establish BUD31 as a critical oncogenic splicing factor with potential as a therapeutic target in ovarian cancer.

What interactions exist between BUD31 and other spliceosome components?

BUD31 forms extensive interactions within the spliceosome complex:

• Protein interaction partners:

  • Immunoprecipitation coupled with mass spectrometry identified 46 annotated spliceosome proteins associated with BUD31

  • BUD31 predominantly interacts with U2 snRNPs and hnRNP proteins

  • In testicular cells, verified interactions include Sf3b1, U2af2, and Ddx4

• Subcellular localization:

  • Immunofluorescence shows BUD31 colocalizes with SC35, a marker of nuclear speckles

  • Nuclear speckles are specialized domains involved in splicing factor storage and assembly

• Functional significance:

  • These interactions position BUD31 as a coordinator of early spliceosome assembly

  • Particularly important for proper splice site recognition and exon definition

Understanding these protein-protein interactions provides mechanistic insight into how BUD31 regulates splicing and suggests potential nodes for therapeutic intervention.

What therapeutic strategies could target BUD31 or its regulated splicing events?

Research suggests multiple approaches for therapeutic targeting of BUD31-mediated splicing:

• Direct BUD31 inhibition:

  • RNAi-based approaches (siRNA, shRNA) show efficacy in preclinical models

  • Inducible knockdown systems demonstrated reduced tumor growth in xenograft models

• Splice-switching antisense oligonucleotides (SSOs):

  • More specific approach targeting individual BUD31-regulated splicing events

  • Demonstrated efficacy with intratumoral injection in mouse models (5 nmol ASO every 3 days)

  • Promotes exon skipping in oncogenic targets like BCL2L12, inducing apoptosis

• Clinical development considerations:

  • Similar approaches for other splicing targets have reached first-in-human trials for glioblastoma

  • Tissue-specific delivery systems would be essential to minimize off-target effects

  • Combination therapies with standard chemotherapeutics might enhance efficacy

These approaches highlight the potential of BUD31-mediated splicing as a novel therapeutic vulnerability in cancer.

How should researchers design experiments to identify novel BUD31 splicing targets?

A comprehensive experimental pipeline for identifying BUD31 targets should include:

• Genome-wide binding site mapping:

  • SpyCLIP or RIP-seq to identify BUD31-bound RNAs

  • Analysis of binding site distribution relative to splice sites

  • Motif discovery using algorithms like HOMER

• Transcriptome-wide splicing analysis:

  • RNA-seq following BUD31 knockdown/knockout

  • Specialized analysis tools to identify different types of alternative splicing events

  • Quantification of percent spliced in (PSI) changes with ≥10% threshold

• Integration of binding and splicing data:

  • Overlap analysis between BUD31-bound genes and alternatively spliced genes

  • In ovarian cancer studies, this approach identified 1,408 direct targets (57% overlap)

• Functional validation:

  • RT-PCR validation of top candidate events

  • Minigene constructs to confirm direct regulation

  • Rescue experiments with wild-type vs. binding-deficient BUD31

• Computational prioritization:

  • Pathway enrichment analysis of targets

  • Prediction of functional consequences (NMD, protein domain disruption)

  • Integration with tissue-specific expression data

This systematic approach enables identification of both direct and indirect BUD31 targets with high confidence.

What are the best animal models for studying BUD31 function in vivo?

Several animal model systems have proven effective for studying BUD31 function:

• Conditional knockout mouse models:

  • Tissue-specific Cre-loxP systems (e.g., Bud31-sKO for germ cell-specific deletion)

  • Allows study of developmental and adult tissue-specific functions

  • Circumvents potential embryonic lethality of complete knockout

• Inducible knockdown systems:

  • Doxycycline-inducible shRNA models (1.2 g/L doxycycline in drinking water)

  • Enables temporal control of BUD31 depletion

  • Useful for studying acute vs. chronic effects

• Xenograft cancer models:

  • Subcutaneous or intraperitoneal injection of cancer cells with BUD31 manipulation

  • Bioluminescence imaging for tracking tumor progression

  • Allows therapeutic intervention testing (e.g., ASO treatment)

• Genetic approaches for mechanistic studies:

  • Splicing reporter mice to monitor BUD31-regulated events in vivo

  • CRISPR-engineered models with mutations in BUD31 binding motifs

  • Compound models combining BUD31 manipulation with target gene alterations

The optimal model depends on the specific research question, with conditional approaches being particularly valuable given BUD31's likely essential functions in development.

How can researchers computationally analyze BUD31-dependent alternative splicing patterns?

Computational analysis of BUD31-dependent splicing requires specialized bioinformatic approaches:

• RNA-seq analysis pipeline:

  • Quality control and read mapping to reference genome

  • Transcript reconstruction and quantification

  • Alternative splicing analysis using tools like rMATS or VAST-TOOLS

  • Classification of event types (exon skipping, intron retention, alternative splice sites)

  • Quantification using metrics like Percent Spliced In (PSI) with IncLevelDifference ≥10%

• CLIP-seq data processing:

  • Peak calling using PURECLIP algorithm for identifying protein-RNA crosslink sites

  • Mapping distribution relative to genomic features (exons, introns, splice sites)

  • De novo motif discovery using HOMER algorithm

  • Integration with RNA-seq data to correlate binding with splicing outcomes

• Functional impact assessment:

  • Analysis of coding sequence (CDS) length changes

  • Prediction of nonsense-mediated decay (NMD) sensitivity

  • Protein domain disruption analysis

  • Pathway enrichment of affected genes (e.g., GO analysis showed enrichment for apoptosis, cell cycle, splicing, and autophagy)

• Visualization techniques:

  • Sashimi plots for individual splicing events

  • Global splicing pattern visualization (heatmaps, volcano plots)

  • Binding site distribution plots relative to splice sites

This multi-dimensional approach provides comprehensive insights into BUD31's splicing regulatory network.

What are the emerging questions about BUD31's role in human disease beyond cancer?

While cancer and reproductive biology have been focal points of BUD31 research, several emerging areas warrant investigation:

• Neurodevelopmental and neurodegenerative implications:

  • Alternative splicing is highly regulated in the nervous system

  • BUD31's impact on exon inclusion could affect neuronal protein diversity

  • Potential connection to splicing-related neurological disorders

• Immune system regulation:

  • Splicing factors play critical roles in immune cell differentiation and function

  • BUD31's regulation of apoptotic pathways may impact immune cell homeostasis

  • Possible involvement in autoimmune conditions through splicing regulation

• Metabolic disorders:

  • Splicing dysregulation is increasingly linked to metabolic syndrome

  • BUD31's targets may include metabolic enzymes and signaling components

  • Tissue-specific splicing programs in liver, muscle, and adipose tissue

• Aging-related processes:

  • Splicing fidelity declines with age across tissues

  • BUD31 expression or function may change during aging

  • Potential contribution to age-related disease susceptibility

Investigation into these areas would extend our understanding of BUD31 beyond current knowledge and potentially identify new therapeutic opportunities.

How might targeting BUD31-regulated splicing advance precision medicine approaches?

BUD31-focused therapeutic strategies offer several advantages for precision medicine:

• Cancer subtype stratification:

  • BUD31 expression varies across cancer types and stages

  • Expression levels correlate with survival outcomes in ovarian cancer

  • Could serve as a biomarker for patient stratification

• Isoform-specific therapeutic targeting:

  • Splice-switching antisense oligonucleotides can target specific BUD31-regulated exons

  • Enables precise modulation of oncogenic splicing events (e.g., BCL2L12 exon 3)

  • Reduces off-target effects compared to direct BUD31 inhibition

• Combination therapy potential:

  • BUD31 inhibition could sensitize resistant tumors to conventional therapies

  • Synergistic effects when combined with other splicing modulators

  • Pathway-specific targeting based on BUD31's splicing program in individual tumors

• Therapeutic resistance mechanisms:

  • Alternative splicing contributes to therapy resistance mechanisms

  • BUD31 inhibition might prevent adaptive splicing changes

  • Sequential or cyclical targeting strategies could prevent resistance development

The highly specific nature of splicing regulation provides opportunities for precise therapeutic intervention with potentially reduced systemic toxicity.

What technological advances will further elucidate BUD31 function?

Emerging technologies will enable deeper insights into BUD31 biology:

• Single-cell splicing analysis:

  • Reveals cell-to-cell heterogeneity in BUD31-regulated splicing

  • Identifies rare cell populations with distinct splicing programs

  • Tracks splicing changes during cellular differentiation or transformation

• Direct RNA sequencing:

  • Long-read sequencing technologies provide full-length transcript information

  • Eliminates PCR and fragmentation biases in splicing detection

  • Enables comprehensive isoform analysis including complex splicing patterns

• Spatial transcriptomics:

  • Maps tissue-specific BUD31 activity and splicing outcomes

  • Reveals microenvironmental influences on BUD31 function

  • Particularly valuable for understanding tumor heterogeneity

• CRISPR screens for splicing modifiers:

  • Identifies genes that synergize with or antagonize BUD31 function

  • Reveals synthetic lethal interactions in cancer contexts

  • Discovers novel therapeutic targets within the splicing regulatory network

These technological advances will provide unprecedented resolution of BUD31's functional impact across diverse biological contexts.