Ladha, F. A. | Search bioRxiv

bioRxiv2025

Dosage-sensitive RBFOX2 autoregulation promotes cardiomyocyte differentiation through transcriptome maturation

Haploinsufficiency of the RNA splicing regulator, RBFOX2, is linked to congenital heart disease (CHD), yet its pathogenic mechanisms remain unclear. Here, we demonstrate that RBFOX2 is essential for progressing cardiomyocyte (CM) differentiation by shifting exon usage profiles to more mature patterns in sarcomere, cytoskeletal, and focal adhesion genes, including alpha-actinin-2 (ACTN2). This maturation program is initiated by critical levels of RBFOX2 that facilitate autoregulatory splicing at mutually exclusive exons encoding early and late isoforms with distinct functional roles. In heterozygous CMs, autoregulation is disrupted, which skews isoform ratios and generates a dominant-negative product caused by exon co-inclusion. Finally, we demonstrate that overexpression of ACTN2 rescues heterozygous, but not null, phenotypes by restoring contractility, which triggers a mechanosensing feedback loop involving upregulation of RBFOX2 from the wildtype allele and transcriptome maturation. Our data suggest that decreased RBFOX2 dosage and autoregulation impair CM differentiation, contributing to CHD pathogenesis and heart failure susceptibility.

developmental biology↗

bioRxiv2020

Identifying dynamic protein and RNA proximity interaction networks of actinin reveals RNA-binding and metabolic regulatory mechanisms

RationaleActinins are actin cross-linkers that are ubiquitously expressed and harbor mutations in heritable diseases. The predominant cardiac actinin is encoded by ACTN2, which is a core structural component of the sarcomere Z-disk. ACTN2 is required for sarcomere function through complex interactions with proteins involved in sarcomere assembly, cell signaling and transcriptional regulation. However, there remain critical gaps in our knowledge of the complete and dynamic cardiac actinin interactome, which could reveal new insights into sarcomere biology. ObjectiveWe sought to examine the cardiac actinin interactome through sarcomere assembly in human cardiomyocytes. Methods and ResultsWe utilized CRISPR/Cas9, induced pluripotent stem cell technology and BioID to reveal cardiac actinin protein interactions in human cardiomyocytes. We identified 324 cardiac actinin proximity partners, analyzed networks, and studied interactome changes associated with sarcomere assembly. We focused additional studies on unexpected actinin interactions with effectors with RNA-binding functions. Using RNA immunoprecipitation followed by sequencing, we determined that RNA-binding partners uncovered by actinin BioID were bound to gene transcripts with electron transport chain and mitochondrial biogenesis functions. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein associated with type 2 diabetes, directly interacted with the rod domain of actinin through its K Homology domain. IGF2BP2 was necessary for electron transport chain transcript localization to vicinal RNA-binding proteins, mitochondrial mass and oxidative metabolism. IGF2BP2 knockdown also impaired sarcomere function in a cardiac microtissue assay. ConclusionsThis study expands our functional knowledge of cardiac actinin, uncovers new sarcomere interaction partners including those regulated by sarcomere assembly, and reveals sarcomere crosstalk with RNA-binding proteins including IGF2BP2 that are important for metabolic functions.

cell biology↗