Abstract 321: RBFOX2 is critical for maintaining alternative polyadenylation patterns in cardiomyoblasts

Abstract

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.