Project Summary: Pulmonary hypertension (PH) is a cardiovascular disorder characterized by high mortality, primarily due to right ventricular (RV) failure (RVF) caused by increased pulmonary vascular resistance. RV fibrosis, a hallmark of decompensated RVF, lacks targeted therapies, highlighting the need to elucidate the molecular mechanisms underlying RV fibrosis and dysfunction. Our research focuses on the role of alternative polyadenylation (APA), a process associated with excessive production of extracellular matrix (ECM) proteins, in RV fibrosis. APA shortens the 3' untranslated region (UTR) of transcripts, leading to loss of microRNA binding sites and increased transcript stability. We have identified Cleavage and Polyadenylation Specific Factor 6 (CPSF6), a key regulator of APA, as being involved in end- stage RVF. In RVF patients, CPSF6 exhibits lengthened 3' UTR and decreased protein expression. Silencing CPSF6 in cardiac fibroblasts (CFs) results in 3' UTR shortening and upregulation of major fibrotic mediators, including TGF-β1 and its receptor, TGFβR1. Pathway analysis further supports 3' UTR shortening in mRNAs encoding ECM proteins in CPSF6 knockdown CFs. Additionally, we have discovered the role of 4-hydroxy-2- nonenal (4HNE), a reactive aldehyde generated during oxidative stress, in RVF. Increased 4HNE downregulates CPSF6, inducing 3' UTR shortening in profibrotic genes and promoting RV fibrosis. The proposed research aims to investigate these mechanisms and identify therapeutic targets for mitigating RV fibrosis. Our hypothesis posits that CPSF6 depletion shortens the 3' UTRs of ECM genes, causing their escape from regulation, promoting their expression, and leading to RV fibrosis. Specifically, we will: Investigate the impact of CPSF6 loss on the 3' UTR landscape and profibrotic gene expression in RVF (Specific Aim 1). Uncover the mechanism underlying CPSF6 reduction-dependent 3' UTR shortening in CFs and its functional consequences in RVF (Specific Aim 2). Assess the impact of ALDH2 restoration on alleviating RV fibrosis through CPSF6 regulation (Specific Aim 3). The validation of our hypotheses and the completion of these aims will highlight the importance of 3' UTR shortening in ECM deposition and fibrosis in RVF, potentially guiding the development of therapeutic interventions. Given the limited treatment options and severe consequences of PH, our research holds significant promise for improving public health.