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.
Dysregulated alternative splicing (AS) that contributes to diabetes pathogenesis has been identified, but little is known about the RNA binding proteins (RBPs) involved. We have previously found that the RBP CELF1 is upregulated in the diabetic heart; however, it is unclear if CELF1 contributes to diabetes-induced AS changes. Utilizing genome wide approaches, we identified extensive changes in AS patterns in Type 1 diabetic (T1D) mouse hearts. We discovered that many aberrantly spliced genes in T1D hearts have CELF1 binding sites. CELF1-regulated AS affects key genes within signaling pathways relevant to diabetes pathogenesis. Disruption of CELF1 binding sites impairs AS regulation by CELF1. In sum, our results indicate that CELF1 target RNAs are aberrantly spliced in the T1D heart leading to abnormal gene expression. These discoveries pave the way for targeting RBPs and their RNA networks as novel therapies for cardiac complications of diabetes.
The RNA binding protein RBFOX2 is linked to heart and skeletal muscle diseases; yet, RBFOX2-regulated RNA networks have not been systematically identified.Although RBFOX2 has a well-known function in alternative splicing (AS), it is unclear whether RBFOX2 has other roles in RNA metabolism that affect gene expression and function. Utilizing state of the art techniques Poly(A)-ClickSeq (PAC-seq) and nanopore cDNA sequencing, we revealed a new role for RBFOX2 in fine tuning alternative polyadenylation (APA) of pre-mRNAs in myoblasts. We found that depletion of RBFOX2 altered expression of mitochondrial genes. We identified the mitochondrial gene Slc25a4 gene that transports ATP/ADP across inner mitochondrial membrane as a target of RBFOX2. Dissecting how RBFOX2 affects Slc25a4 APA uncovered that RBFOX2 binding motifs near the distal polyadenylation site (PAS) are critical for expression of Slc25a4. Consistent with changes in expression of mitochondrial genes, loss of RBFOX2 altered mitochondrial membrane potential and induced mitochondrial swelling. Our results unveiled a novel role for RBFOX2 in maintaining APA decisions and expression of mitochondrial genes in myoblasts relevant to heart diseases. Keywords: alternative polyadenylation/mitochondria/nanopore sequencing/ poly(A) sequencing/RBFOX2 Non-standard Abbreviations and Acronyms: AS alternative splicing PAC-seq poly(A)-ClickSeq APA alternative polyadenylation KD knock down 3´UTR 3´untranslated region PAS poly(A)-site PAC poly(A)-cluster DPAC differential-poly(A)-clustering dPAS distal poly(A)-site pPAS proximal poly(A)-site ANT1 (Slc25a4) ATP/ADP translocator (mitochondrial) MFN1 Mitofusin
Background: The RNA binding protein RBFOX2 is implicated in human heart diseases. However, RBFOX2-regulated RNA networks are not well defined. RBFOX2 has a well-characterized role in alternative splicing (AS) while accumulating evidence suggests that RBFOX2 may also have a role in alternative polyadenylation (APA). Recent studies showed that RBFOX2 binds to regions close to poly(A) sites in the 3’UTR of pre-mRNAs. In addition, RBFOX2 binds to the cleavage and polyadenylation specificity factors. Therefore, we aimed to determine whether RBFOX2 has a role in regulating APA. Method: We employed poly(A)click sequencing (PAC-seq) and DPAC (Differential Poly(A) Cluster analysis) computational pipeline to identify differential poly(A) usage and mRNA abundance. We also used nanopore sequencing to identify different spliced variants and the coordinated AS and APA events. Results: We report that knockdown of RBFOX2 in embryonic rat heart derived cells leads to altered alternative polyadenylation (APA) of hundreds of genes. RBFOX2-mediated APA changes impacted both mRNA levels and generation of different gene isoforms. Nanopore sequencing identified full-length transcripts regulated by RBFOX2 and revealed RBFOX2-mediated isoform switches via both APA and AS in cardiac cells. Notably, RBFOX2-regulated APA networks affect genes such as Tpm1 and Tnnt1 involved in cardiac contractility. Identification of RBFOX2-regulated RNA networks provides novel insights into the pathogenesis of heart diseases in which RBFOX2 is involved and pave the way for designing therapeutics. Conclusions: RBFOX2 regulates alternative polyadenylation via splicing dependent and independent mechanisms. RBFOX2-mediated APA affects mRNA levels of contractile genes.
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