RBM39 and Its Role in RNA Splicing, Function, and Disease

RNA-binding motif protein 39 (RBM39) plays a key role in RNA processing, particularly in alternative splicing. As part of the broader family of RNA-binding proteins, it regulates gene expression by influencing how precursor mRNA is processed into mature transcripts. Given that precise splicing is crucial for cellular function, RBM39’s activity has significant implications for both normal physiology and disease.

Dysregulation of RBM39 has been linked to various cancers and other disorders, making it an important subject of biomedical research. Understanding its molecular interactions and regulatory mechanisms provides insight into fundamental biology and potential therapeutic strategies.

Cellular Role And Key Functions

RBM39 regulates pre-mRNA splicing by binding to specific RNA sequences, influencing exon inclusion or exclusion. This function generates protein isoforms with distinct roles, particularly in tissues with high transcriptional activity, such as hematopoietic and epithelial cells. RBM39 interacts with splicing factors like U2AF65, reinforcing its role in splice site recognition and alternative exon usage.

Beyond splicing, RBM39 stabilizes certain transcripts, preventing degradation and ensuring availability for translation. This function is relevant in pathways regulating the cell cycle and apoptosis. Research has shown that RBM39 influences the expression of genes involved in DNA damage response by modulating splicing and mRNA stability.

Its activity varies across cell types and physiological conditions. During embryonic development, it regulates splicing programs essential for lineage specification, ensuring progenitor cells generate the correct protein isoforms. In adult tissues, it maintains homeostasis by adjusting splicing patterns in response to environmental cues such as stress or hormonal signaling. This adaptability allows cells to dynamically regulate their transcriptomes.

Structural Domains

RBM39 contains two RNA recognition motifs (RRMs) that mediate interactions with RNA sequences. These domains adopt a conserved β-sheet and α-helix arrangement, forming a platform for recognizing nucleotide motifs in pre-mRNA. Structural studies show that RBM39’s RRMs exhibit sequence preferences that guide alternative splicing by influencing exon inclusion or exclusion.

Beyond the RRMs, RBM39 has a ubiquitin-associated (UBA)-like domain that facilitates interactions with splicing factors, stabilizing complexes involved in spliceosome assembly. This domain enables RBM39 to act as a scaffold, bringing together RNA substrates and regulatory proteins. Proteomic analyses have identified RBM39 in splicing-associated complexes with U2AF65 and SF3B1, highlighting its role in coordinating splice site selection.

The C-terminal region functions as an intrinsically disordered domain (IDD), contributing to regulatory flexibility. This segment allows RBM39 to engage in transient interactions with multiple splicing regulators. Emerging evidence suggests RBM39 may participate in ribonucleoprotein granules that concentrate splicing factors at specific nuclear sites, enhancing spliceosome assembly efficiency.

Links To Splicing Machinery

RBM39 influences splicing through direct interactions with spliceosome components. It associates with U2AF65, a splicing factor that facilitates recognition of the polypyrimidine tract near 3′ splice sites. By binding U2AF65, RBM39 enhances U2 snRNP recruitment, a critical step in spliceosome assembly. Structural analyses show that RBM39 stabilizes U2AF65’s RNA-binding conformation, fine-tuning its affinity for pre-mRNA targets.

RBM39 also interacts with SF3B1, a component of the U2 snRNP complex that facilitates branch point recognition. SF3B1 mutations are common in myelodysplastic syndromes and certain tumors, and RBM39 may modulate both wild-type and mutant SF3B1 activity. By engaging with SF3B1, RBM39 helps recruit the spliceosome’s catalytic core, ensuring efficient intron excision.

Additionally, RBM39 contributes to the assembly of early spliceosomal complexes, specifically the E and A complexes, which establish the foundation for subsequent catalytic steps. Through its RNA recognition motifs and protein-protein interaction domains, RBM39 positions splicing factors correctly, preventing exon skipping or intron retention. Its involvement in alternative splicing programs allows dynamic transcript regulation in response to cellular cues.

Observed Mutations And Dysregulation

Genomic alterations in RBM39 have been identified in various cancers and developmental disorders, often disrupting normal splicing patterns. Missense mutations within its RNA recognition motifs can alter binding affinity for pre-mRNA, leading to aberrant exon inclusion or skipping. Certain leukemia subtypes exhibit RBM39 mutations that favor splice variants promoting unchecked cell proliferation. Structural analyses show that these mutations reduce RBM39’s stability and interactions with splicing cofactors, compounding splicing errors.

Beyond genetic mutations, RBM39 is frequently dysregulated through post-translational modifications and altered expression levels. Increased RBM39 expression has been observed in breast and ovarian cancers, where it enhances exon inclusion linked to tumor growth. Conversely, RBM39 degradation can be triggered by small-molecule inhibitors like indisulam, which recruits RBM39 to the DCAF15 E3 ubiquitin ligase complex for proteasomal degradation. This targeted depletion disrupts splicing programs essential for cancer cell survival, highlighting RBM39 as a therapeutic target.

Associations With Disease

Aberrant RBM39 expression and function have been implicated in cancers where splicing dysregulation contributes to tumor progression. Elevated RBM39 levels have been observed in acute myeloid leukemia (AML), breast cancer, and ovarian cancer, where it promotes exon inclusion that enhances cell survival and proliferation. In AML, increased RBM39 expression alters the splicing of apoptosis-related genes, leading to resistance against programmed cell death. Targeting RBM39 with small-molecule degraders like indisulam induces widespread intron retention and exon skipping, selectively impairing cancer cell viability.

Beyond cancer, RBM39 has been associated with neurodevelopmental disorders, where splicing disruptions impair neuronal differentiation and synaptic function. Mutations in RBM39 or its interacting partners have been linked to developmental delay and intellectual disability, likely due to misprocessing of transcripts essential for neural circuit formation. Transcriptomic analyses suggest RBM39 dysfunction may also play a role in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where splicing defects lead to toxic RNA accumulation. These findings highlight RBM39 as a key regulator of cellular homeostasis, with its misregulation contributing to both malignant and degenerative diseases.

Research Techniques For Studying RBM39

Investigating RBM39 function requires molecular and genomic techniques to dissect its role in RNA processing. Advances in gene-editing, immunoprecipitation, and sequencing technologies have provided tools to analyze RBM39’s interactions, splicing targets, and regulatory mechanisms.

CRISPR-Based Knockdowns

CRISPR-Cas9 and CRISPR interference (CRISPRi) have been used to reduce RBM39 expression and assess its impact on gene regulation. CRISPRi, which uses a catalytically inactive Cas9 fused to a transcriptional repressor, enables precise downregulation without inducing double-strand breaks. Functional studies show RBM39 depletion leads to widespread exon skipping and intron retention, particularly in transcripts involved in cell cycle control. These knockdown models have validated RBM39 as a therapeutic target, as cells lacking RBM39 exhibit increased sensitivity to splicing modulators.

RNA Immunoprecipitation Analysis

RNA immunoprecipitation (RIP) combined with quantitative PCR or sequencing (RIP-seq) identifies RBM39-bound transcripts. By crosslinking RBM39 to its RNA targets, researchers isolate and sequence associated RNA molecules, revealing its splicing preferences and regulatory networks. This technique has shown RBM39 preferentially binds pre-mRNAs with weak splice sites, reinforcing its role in alternative exon inclusion. RIP analyses have also uncovered interactions between RBM39 and long non-coding RNAs (lncRNAs), suggesting its function extends beyond conventional pre-mRNA splicing to broader RNA metabolism.

Next-Generation Sequencing Approaches

RNA sequencing (RNA-seq) and specialized variants, such as long-read sequencing and splicing-sensitive assays, provide high-resolution insights into RBM39-dependent splicing events. Differential splicing analysis following RBM39 depletion has identified hundreds of misregulated exons, many enriched in genes controlling apoptosis and DNA repair. Long-read sequencing has further refined the understanding of isoform diversity, revealing that RBM39 regulates not only exon inclusion but also alternative 3′ and 5′ splice site selection. These sequencing strategies have mapped the full extent of RBM39’s influence on transcriptome dynamics, offering a comprehensive view of its role in gene expression regulation.