RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Abstract

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome

“…Consistent with this finding, a previous report documented decreased expression of sarcomere and mitochondrial components in Rbfox2-depleted rat myoblasts due to alternative polyadenylation 33 . However, conditional knock-out of Rbfox2 in the embryonic mouse heart was linked to changes in alternative splicing of transcripts involved in cell adhesion to the extra-cellular matrix 14 , a process not readily identified in our datasets. Therefore, a more comprehensive cross-species comparison of alternative splicing changes caused by loss of Rbfox2 in the heart could be informative.…”

“…Arguing against pathogenicity, mice with Rbfox2 deleted in Mlc2v + cardiomyocytes are born with grossly normal hearts 13 . However, a recent report demonstrated that earlier deletion of Rbfox2 in Nkx2.5 + cells, which include cardiac progenitors, is embryonic lethal by E11.5 14 . Specifically, these animals are severely growth impeded and display gross morphological defects in cardiac chamber, common outflow tract, and yolk sac vasculature development.…”

FOXD1 Is a Transcription Factor Important for Uveal Melanocyte Development and Associated with High-Risk Uveal Melanoma

“…We identified the upregulation of two transcription regulators, RBFOX2 and FOXD1 , in high-risk UM. RBFOX2 is known to be involved in cerebellar and heart development [ 49 , 50 ], and FOXD1 is involved in retina development [ 51 , 52 ], but to our knowledge neither have been associated with melanocyte development prior to this study. RBFOX2 is a well-known regulator of epithelial-to-mesenchymal transition [ 53 ], where the alternate splicing of pre-mRNA alters signal transduction via a wide range of genes depending on cellular context [ 54 , 55 , 56 ].…”

Post-transcriptional control by RNA-binding proteins in diabetes and its related complications

“…Previous studies showed that CELF1 is up-regulating in the hearts of T1DM mice, but diabetes-induced AS alterations are consistent with CELF1 depletion or decreased CELF1 splicing activity ( Blech-Hermoni et al, 2016 ; Belanger et al, 2018 ). Interestingly, RBFox2, an RNA-binding protein belonging to the RBFOX family, that is involved in AS regulation in heart diseases, shows the same trend as CELF1 ( Gazzara et al, 2017 ; Verma et al, 2022 ). RBFox2 regulates cardiac function-related genes associated with diabetic cardiomyopathy.…”

“…RBPs can regulate pre-mRNA alternative splicing (AS) through binding to the pre-mRNA and interacting with the spliceosome components, which generate variant protein isoforms from a single gene, resulting in transcriptome and proteome diversity ( Sperling, 2017 ). Previous studies demonstrated that AS controlled by RBPs plays a crucial role in diabetes and its complications ( Verma et al, 2013 ; Nutter et al, 2016 ; Gazzara et al, 2017 ; Belanger et al, 2019 ; Verma et al, 2022 ). The role of RBPs in AS will be discussed in detail in the next section.…”