PROJECT SUMMARY Although impressive progress has been made to reduce the burden of heart disease, the illness remains the leading cause of death in the United States. Notably, many types of persistent heart injury can cause pathological remodeling of the heart and progression to heart failure (HF). Our goal is to identify novel molecular pathways responsible for such transition, since targeting them therapeutically presents an exciting opportunity to reverse heart remodeling and improve survival. While transcriptional regulators of progression to HF have been studied extensively, the importance of post-transcriptional regulation, such as chemical modification of messenger RNA, has been overlooked. Recent studies recognized that methylation of mRNA in position N6 of adenosines (m6A) was essential for the heart’s ability to adapt to stress, but the master regulator of this mechanism remains unknown. This proposal is aimed to identify the undiscovered cardiac mediator of m6A-dependent mechanism and elucidate how exactly m6A methylation acts to maintain homeostasis in the heart. After methylation, m6A- modified mRNAs can be recognized by specific mRNA-binding proteins belonging to the YTH domain family (YTHDF), of which YTHDF1 is a key member expressed in the heart. Our preliminary proteomics data from mouse hearts show that YTHDF1 is the only member in its family to interact with the protein Hook homolog 3 (HOOK3), which binds motor proteins to activate cargo movement along the microtubules. Since ribonucleoproteins can use microtubular routes to reach their sites of localized translation, our central hypothesis is that YTHDF1 is critical for maintenance of cardiac homeostasis post-stress by modulating m6A-mRNA translation through its interaction with HOOK3. To test this hypothesis, we have already generated a novel conditional mouse model with cardiomyocyte-specific deletion of YTHDF1. In Aim 1, we will subject control and cardiomyocyte-specific YTHDF1 knockout mice to a pressure-overload model of heart failure to define the requirement of YTHDF1 for cardiac stress adaptation. In Aim 2, we will then perform ribosome profiling to determine the global impact of YTHDF1 on mRNA translation and crosslinking immunoprecipitation assays to identify its key mRNA targets in adult cardiomyocytes. Finally, in Aim 3 we will characterize YTHDF1 interaction with HOOK3 and analyze its functional significance using a variety of exciting molecular biology techniques. Overall, our approach is innovative because it aims to define the entirely novel function of YTHDF1 in the heart. This proposal is also significant because it will fill knowledge gaps about the mechanism for YTHDF1-dependent regulation of m6A-mRNA fate through our newly discovered HOOK3 interaction. The proposed study is also significant because targeting direct effectors of mRNA processing such as YTHDF1 opens new avenues for correcting pathological protein synthesis in the heart. Markedly, such knowle...