RNA splicing factor RBFOX2 is a key factor in the progression of cancer and cardiomyopathy

Abstract

Background: Alternative splicing of pre-mRNA is a fundamental regulatory process in multicellular eukaryotes, significantly contributing to the diversification of the human proteome. RNA-binding fox-1 homologue 2 (RBFOX2), a member of the evolutionarily conserved RBFOX family, has emerged as a critical splicing regulator, playing a pivotal role in the alternative splicing of pre-mRNA. This review provides a comprehensive analysis of RBFOX2, elucidating its splicing activity through direct and indirect binding mechanisms. RBFOX2 exerts substantial influence over the alternative splicing of numerous transcripts, thereby shaping essential cellular processes such as differentiation and development.

Main body of the abstract: Dysregulation of RBFOX2-mediated alternative splicing has been closely linked to a spectrum of cardiovascular diseases and malignant tumours, underscoring its potential as a therapeutic target. Despite significant progress, current research faces notable challenges. The complete structural characterisation of RBFOX2 remains elusive, limiting in-depth exploration beyond its RNA-recognition motif. Furthermore, the scarcity of studies focusing on RBFOX2-targeting drugs poses a hindrance to translating research findings into clinical applications.

Conclusion: This review critically assesses the existing body of knowledge on RBFOX2, highlighting research gaps and limitations. By delineating these areas, this analysis not only serves as a foundational reference for future studies but also provides strategic insights for bridging these gaps. Addressing these challenges will be instrumental in unlocking the full therapeutic potential of RBFOX2, paving the way for innovative and effective treatments in various diseases.

Keywords: RBFOX2; alternative splicing; pre‐mRNA; therapeutic targeting.

FIGURE 1

Decoding RNA‐binding fox‐1 homologue 2 (RBFOX2): insights into structure, function and RNA‐binding conformation. (A) Primary structure of RBFOX2: the protein sequence of RBFOX2 is compared with its family proteins RBFOX1 and RBFOX3. Notably, the sequences of the RNA‐recognition motif (RRM) and nuclear localisation signal (NLS) are identical in RBFOX2 and RBFOX1. (B) Comparison of RRM between RBFOX2 and RBFOX1: a detailed comparison of the RRM between RBFOX2 and RBFOX1 highlights their structural similarities. (C) RNA‐binding conformation of RBFOX1 with UGCAUGU motif: the RNA‐binding conformation of RBFOX1 with the UGCAUGU motif is depicted. Within this motif, Phe126 plays a crucial role in binding U1, G2, C3 and A4, while His120, Phe158 and Phe160 are essential for binding U5, G6 and U7. Protein structure data utilised in this analysis is sourced from the Protein Data Bank (2CQ3 and 2ERR) and visualised using ChimeraX. (D) Various transcripts of human RBFOX2 depict a complex exon organisation, where constitutive exons are delineated by orange boxes, alternative exons by grey‒blue boxes, and regions potentially untranslated by red gradients. Exon numbering serves for precise identification, correlating with genomic coordinates detailed in Table S1. We numbered the RBFOX2 exons with Arabic numerals based on the order of their starting sites on the chromosome. Alternative exons were numbered with letters according to their starting sites on the chromosome. This splicing procedure ensures that each exon has a unique identifier, allowing for a clearer description of the gene structure and alternative splicing forms. It is important to note that this exon numbering may not align with previously reported exon designations (coordinate information of RBFOX2 exons based on GRCh38/hg38).

FIGURE 2

RNA‐binding fox‐1 homologue 2 (RBFOX2) splicing modes: direct and indirect binding mechanisms unraveled. In the direct binding mode, RBFOX2 specifically recognises target RNA sequences containing the (U)GCAUG motif through its inherently conserved RNA‐recognition motif (RRM). When RBFOX2 binds upstream of a target exon splice site, it consistently leads to exon skipping. Conversely, binding downstream of the splice site results in exon inclusion. In the indirect binding mode, RBFOX2 interacts with target RNA indirectly by binding to other RNA‐binding proteins (RBPs). This interaction enables RBFOX2 to cross‐link with the target RNA, allowing it to exert splicing activity regardless of whether the RNA sequence contains the complete (U)GCAUG motif or not. PBD, protein‐binding domain; RBD, RNA‐binding domain; RBP, RNA‐binding protein; RRM, RNA‐recognition motif. Created with BioRender.com.

FIGURE 3

RNA‐binding fox‐1 homologue 2 (RBFOX2): orchestrating cardiomyocyte function. RBFOX2 plays a pivotal role in regulating physiological mechanisms closely associated with cardiomyocyte function, such as excitation‒contraction coupling (E‒C coupling), calcium ion channels and mitochondrial metabolism, by influencing the alternative splicing of downstream genes. Dysregulation of RBFOX2 has been implicated in the onset of various cardiovascular diseases, including arrhythmias and cardiomyopathy. Specifically, RBFOX2 can promote the expression of Jph2 by inhibiting the maturation of miR‐34a, thereby enhancing the conduction of electrical signals in cardiomyocytes. RBFOX2 also inhibits the inclusion of Cacna1c exon 9′, thereby stabilising the number of L‐type calcium channels in cardiomyocyte membranes and promoting calcium transients after depolarisation. Additionally, RBFOX2 promotes the inclusion of MICU1 exon 5′, enhancing mitochondrial Ca²⁺ uptake and thereby boosting energy metabolism. RBFOX2 also facilitates the inclusion of Enah exon 13, Sorbs2 exon 4 and Tpm1 exon 9b, while inhibiting the inclusion of Tpm1 exon 6a, thus promoting muscle fibre contraction. EI, exon inclusion; EMG, electromyographic; ES, exon skipping. Created with BioRender.com.

FIGURE 4

RNA‐binding fox‐1 homologue 2 (RBFOX2): master regulator of cancer cell behaviour. RBFOX2 exerts control over the biological behaviours of diverse cancer cells, including proliferation, epithelial‒mesenchymal transition (EMT), invasion and migration. It achieves this by directly or indirectly modulating the alternative splicing of genes associated with cancer. Imbalances in RBFOX2 expression are implicated in the onset and progression of cancers across various systems, including digestive, respiratory and haematological systems. BrC, breast cancer; CRC, colorectal cancer; EI, exon inclusion; ES, exon skipping; GBM, glioblastoma multiforme; GC, gastric cancer; NPC, nasopharyngeal carcinoma; pAML, paediatric acute myeloid leukaemia; PDAC, pancreatic ductal adenocarcinoma. Created with BioRender.com.

FIGURE 5

RNA‐PROTAC: targeted degradation strategy extending to RBFOX proteins. RNA‐PROTAC consists of a genetically encoded RNA scaffold (containing an aptamer, linker and a RNA consensus binding element [RCBE]) and a synthetic bifunctional small molecule (one end binds the RNA scaffold and the other end binds E3 ubiquitin ligase). When the RCBE of RNA‐PROTAC contains the RNA‐binding fox‐1 homologue 1 (RBFOX1) binding sequence (UGCAUGU), RBFOX1 can bind to the RNA scaffold of the RNA‐PROTAC. At the same time, the bifunctional small molecules on RNA‐PROTAC can recruit E3 ubiquitin ligase to the RNA scaffold in a non‐covalent manner. The recruited E3 ubiquitin ligase specifically induces the ubiquitination of the target protein (i.e., RBFOX1) and its degradation through the ubiquitin‒proteasome pathway. This targeting method is also expected to be extended to RBFOX2. PROTAC, proteolysis targeting chimera; RCBE, RNA consensus binding element; Ub, ubiquitin. Created with BioRender.com.