Abstract Epigenetic regulators are crucial in regulating normal hematopoiesis and are frequently mutated in hematopoietic diseases. Recent genome-wide analyses in samples from MDS patients revealed that epigenetic regulators and splicing factors are frequent mutational targets. Seminal studies published by our lab demonstrated that conditional loss of de novo DNA methyltransferase 3A (Dnmt3a) leads to dramatic self-renewal and inhibited differentiation in hematopoietic stem cells (HSCs). DNMT3A is mutated in 10-13% of MDS patients and is the most frequent mutational target in clonal hematopoiesis. Our preliminary data suggest that DNMT3A directly impacts RNA splicing in the hematopoietic system through interaction with spliceosome-associated factors. This novel finding is significant, as the role of DNMT3A in maintaining normal hematopoiesis has not been extensively studied beyond DNA methylation. The overarching objective of this study is to delineate the functional role of DNMT3A in RNA splicing and determine how DNMT3A-deficient HSCs disrupt normal hematopoiesis. We will test the hypothesis that DNMT3A influences the RNA splicing program in hematopoietic stem and progenitor cells (HSPCs) and may contribute to MDS pathogenesis in part through loss of splicing fidelity. Using co- immunoprecipitation assays, nucleic acid treatments, and pathogenic DNMT3A mutants, we will investigate the mechanistic basis of the interaction between DNMT3A and splicing factors. Additionally, we will delineate the role of DNMT3A in RNA splicing through in vivo deletion of DNMT3A in human cord blood HSPCs using CRISPR/Cas9 strategies. We will identify key groups of genes that are differentially spliced after DNMT3A knockout through deep RNA sequencing. We will also perform colony-forming assays with DNMT3A knockout HSPCs in the presence of H3B-8800, a splicing inhibitor that has recently gone into clinical trials. Additionally, analysis of RNA-sequencing data from Dnmt3a KO HSCs showed aberrant splicing patterns compared to WT HSCs. Therefore, we will attempt to rescue these aberrant splicing patterns through an inducible system that re- expresses DNMT3A in mouse HSCs. These studies will reveal the functional role of DNMT3A in RNA splicing, increasing our understanding of the mechanisms that govern normal hematopoiesis. As current therapies for MDS are ineffective, delineating DNMT3A's role in splicing may provide an avenue for developing novel therapeutic targets to combat this disease.