PROJECT SUMMARY Proper erythropoiesis requires coordinated changes in gene expression and protein synthesis for stem and progenitor cells to differentiate and amass the striking amount of iron-laden hemoglobin required to sustain life in humans. Anemia?insufficient circulating erythrocytes?is most frequently caused by iron deficiency which remains one the most common nutritional deficit in the world. Beyond hemoglobin production, the role of iron in other cellular processes during erythropoiesis, such as gene expression and RNA processing is poorly understood. RNA processing events are mediated by RNA binding proteins, such as the poly C binding proteins (PCBPs). PCBP2 regulates erythropoiesis via alternative splicing of gene transcripts that drive erythroid differentiation programs. Surprisingly, PCBPs were also discovered to function as intracellular iron chaperones, raising the question of whether iron-binding occurs independently of or in concert with RNA-binding. Given that iron levels intimately modulate red blood cell differentiation, we sought to investigate whether PCBP activity is modulated by iron in the context of red cell development. Specifically, we hypothesize that iron modulates the affinity or specificity of PCBPs towards target RNAs causing changes in RNA splicing that are important for red blood cell differentiation. We will address this hypothesis by i) determining the effect of iron on PCBP-RNA interactions using unbiased high throughput in vitro assays, ii) evaluating the effect of iron on PCBP-regulated RNA splicing events in cells, and iii) determine global gene expression and RNA splicing changes in primary human and mouse samples with and without iron deficiency. From these experiments we will uncover iron- sensitive RNA splicing programs that dictate erythropoiesis.