PROJECT SUMMARY The relatively finite number of ~20,000 protein coding genes in the mammalian genome limits the overall diversity that transcriptional regulation can achieve in a given biological context. This diversity can be enhanced by many orders of magnitude by post-transcriptional regulation such as alternative splicing and modification and degradation of mRNA, which in turn leads to a greater protein diversity and thus a greater functional complexity of the cell. This seems particularly relevant in the context of complex processes such as cell specification and organogenesis. The importance of RNA regulation is further supported by the large number RNA-binding proteins encoded in the human genome (~1,500) and their ever more prominent emergence in the context of disease. Despite the clear evidence of the relevance of RNA regulation, much less remains known compared to the exceptionally well-researched gene regulatory mechanisms. This lack of progress is in part due to technical challenges associated with studying and manipulating RNA, particularly during early development. However, recent advancements in this area now afford new opportunities to broaden our knowledge in these critical regulatory mechanisms and start to fill this critical knowledge gap. Here we will interrogate RNA-binding proteins important for cardiac development, specifically the the X-linked protein DDX3X. Mutations in DDX3X lead to DDX3X Syndrome, which is characterized by intellectual disability, autism spectrum disorder, congenital brain malformations and motor problems. DDX3X has not been interrogated during cardiogenesis to date, but recent studies have uncovered that individuals with DDX3X Syndrome frequently present with congenital heart disease. Based on our preliminary findings we hypothesize that DDX3X acts in a spatio-temporal manner, by targeting distinct regulatory networks during the development of the different cell types of the heart. We further hypothesize that disease-causing mutations in DDX3X affect cellular dysfunction by specific mechanisms of DDX3X target regulation. Aim 1 will uncover the direct, and functionally relevant targets of DDX3X. Aim 2 will determine the cellular phenotype of DDX3X-deficient male and female embryos. Aim 3 will elucidate genotype-phenotype correlations in DDX3X Syndrome patient-derived hPSC-CMs. Successful completion of the aims are expected to identify and characterize the cellular requirements and in depth molecular mechanisms of DDX3X, a new RNA-binding protein that we found to be essential for heart development. The overall and long-term goal is to contribute to a better understanding of RNA regulatory mechanisms, specifically during cardiac development and in the context of the formation of congenital heart disease.