Abstract Body: Background: RBFOX2 is an RNA binding protein that regulates alternative splicing (AS) of pre-mRNA transcripts and in turn, generation of different mRNA and protein isoforms. RBFOX2 is implicated in adult and congenital heart diseases. Dysregulation of RBFOX2 is linked to diabetic cardiomyopathy characterized by a dilated left ventricle and stiffening of the heart. Mutations in RBFOX2 leads to Hypoplastic Left Heart Syndrome, a congenital heart disease characterized by hypoplasia of the left ventricle, aorta, and cardiac valves. Interestingly, RBFOX2 loss-of-function mediated AS changes affect different genes and processes in adult vs congenital heart disease. Furthermore, conditional Rbfox2 knockout mice display defective endothelial/endocardial cell mesenchymal transition, adhesion to extracellular matrix (ECM), and migration. Despite RBFOX2’s prominent role in heart diseases, it is unknown how RBFOX2 loss of function in adult vs embryonic stages results in distinct heart diseases. We have also found that RBFOX2 loss affects focal adhesion structures that could be sensing the differences between the stiff ECM in a fibrotic cardiomyopathy heart and a soft cardiac ECM of a developing heart. Based on these findings, we hypothesize that surface stiffness is driving RBFOX2 differential splicing in fetal vs adult hearts.
Hypothesis: We hypothesize that changes in substrate stiffness create signaling cascades that ultimately modify RBFOX2 to affect binding dynamics.
Methods/Results: We performed RNA-seq on RBFOX2 depleted cells grown on a soft vs stiff surface. AS analysis using RMATs algorithm revealed unique RBFOX2-dependent splicing signatures on either surface (False Discovery Rate (FDR) <0.05, |InclusionLevelDifference| ≥0.15). Preliminary results from 2-dimensional gel electrophoresis show a shift in RBFOX2 isoelectric point on different surfaces indicative of post-translational modifications. Our ongoing work is characterizing modifications on RBFOX2 protein and elucidating differential interactions on a soft vs stiff surface.
Conclusions: Our results suggest that RBFOX2 is influenced by changes in surface stiffness. Changes in RBFOX2-dependent splicing programs are representative of fetal and adult heart diseases. Ongoing work will identify specific mechanisms of RBFOX2 regulation and specific AS events that may be targetable for therapeutics in HLHS and diabetic heart disease.