Abstract P2059: Poly(rC)-binding Protein-1 (Pcbp1) Is Essential For Heart Development

Abstract

Rationale: Proper development of the heart relies on a tightly regulated, yet diverse, pattern of gene expression that is governed by transcriptional, post-transcriptional, and translational processes. Congenital heart disease (CHD) is the most common birth defect and a leading cause of morbidity and mortality in children. While the genetic cause of some types of CHD has been identified, the molecular basis for the rest remains elusive. Poly(rC)-binding protein 1 (Pcbp1) is a RNA-binding protein that regulates RNA processing as well as post-transcriptional and translational processes in a variety of biological systems. Hypothesis: Pcbp1 play a critical role in regulating heart development by governing Notch and UPR pathways and mediating proper Aars2 gene splicing. Methods and Results: Germline deletion of Pcbp1 results in lethality before embryonic day (E) 8.5. We generated a cardiac-specific deletion of Pcbp1 by crossing Pcbp1-Flox with cTNT-Cre mice (Pcbp1-cKO cTNT ) and found that Pcbp1-cKO cTNT mice is 50% lethal perinatally. Embryonic hearts of Pcbp1-cKO cTNT mice displayed ventricular non-compaction and abnormal ventricular apex formation. Deep RNA sequencing of Pcbp1-cKO cTNT hearts revealed alteration of gene expression profiles reflective on ventricular maturation delay and dysregulation of Notch and UPR pathways. Interestingly, loss of Pcbp1 in cardiomyocytes disrupts alternative splicing of many important genes including Aars2, a gene associated with congenital mitochondrial cardiomyopathy. Pcbp1 deficiency resulted in creation of an Aars2 exon16-skipping variant, leading to its premature termination. eCLIP-seq showed that Pcbp1 primarily binds to CU-rich motifs at 3’UTR, distal intron and CDS regions of targets, and it interacts with regions of Aars2 transcript near exon 16. Using CRISPR/Cas9 technology, we knocked in loxP sites flanking the exon 16 of Aars2 (Aars2-Flox), and cross the floxed mice with cTNT-Cre mice to generate cardiac-specific exon16 deletion mutant of Aars2 (Aars2-cKO cTNT ). Intriguingly, abnormality in Aars2-cKO cTNT embryonic heart phenocopy aspects of ventricular non-compaction and malformation observed in Pcbp1-cKO cTNT heart. Accordingly, the transcriptome from hearts of Pcbp1-cKO cTNT and Aars2-cKO cTNT display high concordance and similarity and share strikingly common dysregulated pathways. Conclusions: We discover a novel function of Pcbp1 in heart development by regulating Notch and UPR pathways. Additionally, Pcbp1 is indispensable for Aars2 gene splicing, whose deficiency is associated with congenital cardiomyopathy. Our findings suggest modulating Pcbp1 in developing hearts may offer a novel therapeutic intervention for congenital heart failure.